Immunomodulatory proteins with tunable affinities

ABSTRACT

Provided are immunomodulatory proteins and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of InternationalApplication No. PCT/US2016/027995, filed Apr. 15, 2016, which claimspriority from U.S. provisional application No. 62/149,437 filed Apr. 17,2015, entitled “Immunomodulatory Proteins with Tunable Affinities” andto U.S. provisional application No. 62/218,534 filed Sep. 14, 2015,entitled “Immunomodulatory Proteins with Tunable Affinities,” thecontents of each of which are incorporated by reference in theirentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled761612000100_SeqList.txt, created Oct. 16, 2017 which is 377 kilobytesin size. The information in the electronic format of the SequenceListing is incorporated by reference in its entirety.

FIELD

The present invention relates to therapeutic proteins for modulatingimmune response in the treatment of cancer and immunological diseases.

BACKGROUND

Modulation of the immune response by intervening in the processes thatoccur in the immunological synapse (IS) formed by and betweenantigen-presenting cells (APCs) or target cells and lymphocytes is ofincreasing medical interest. Currently, biologics used to enhance orsuppress immune response have generally been limited to immunoglobulins(e.g., anti-PD-1 mAbs) or soluble receptors (e.g., Fc-CTLA4). Solublereceptors suffer from a number of deficiencies. While useful forantagonizing interactions between proteins, soluble receptors often lackthe ability to agonize such interactions. Antibodies have proven lesslimited in this regard and examples of both agonistic and antagonisticantibodies are known in the art. Nevertheless, both soluble receptorsand antibodies lack important attributes that are critical to functionin the IS. Mechanistically, cell surface proteins in the IS can involvethe coordinated and often simultaneous interaction of multiple proteintargets with a single protein to which they bind. IS interactions occurin close association with the junction of two cells, and a singleprotein in this structure can interact with both a protein on the samecell (cis) as well as a protein on the associated cell (trans), likelyat the same time. Thus, there is a need for improved molecules formodulation of immune responses. Provided are embodiments that meet suchneeds.

SUMMARY

Provided herein are immunomodulatory proteins that exhibit alteredbinding affinities to binding partners that are immune protein ligandsinvolved in immunological responses. In some embodiments, the providedimmunomodulatory proteins can modulate, such as potentiate or dampen,the activity of the immune protein ligand, thereby modulatingimmunological responses. In some embodiments, also provided are methodsand uses for modulating immune responses by contacting cells expressingone or more of the immune proteins ligands with a providedimmunomodulatory protein, for example, in the immunotherapeutictreatment of diseases or conditions that are treatable by modulating ofthe immune response.

In some embodiments, provided herein is an immunomodulatory protein,comprising at least one non-immunoglobulin affinity modifiedimmunoglobulin superfamily (IgSF) domain comprising one or more aminoacid substitution(s) in a wild-type IgSF domain, wherein: the at leastone affinity modified IgSF domain has increased binding to at least twocognate binding partners compared to the wild-type IgSF domain; and theat least one affinity modified IgSF domain specifically bindsnon-competitively to the at least two cognate binding partners. In someembodiments, the at least two cognate binding partners are cell surfacemolecular species expressed on the surface of a mammalian cell. In someembodiments, the cell surface molecular species are expressed in cisconfiguration or trans configuration. In some embodiments, the mammaliancell is one of two mammalian cells forming an immunological synapse (IS)and each of the cell surface molecular species is expressed on at leastone of the two mammalian cells forming the IS. In some embodiments, atleast one of the mammalian cells is a lymphocyte. In some embodiments,the lymphocyte is an NK cell or a T cell. In some embodiments, bindingof the affinity modified IgSF domain modulates immunological activity ofthe lymphocyte.

In some embodiments, the immunomodulatory protein is capable ofeffecting increased immunological activity compared the wild-typeprotein comprising the wild-type IgSF domain. In some embodiments, theimmunomodulatory protein is capable of effecting decreased immunologicalactivity compared to the wild-type protein comprising the wild-type IgSFdomain. In some embodiments, at least one of the mammalian cells is atumor cell. In some embodiments, the mammalian cells are human cells. Insome embodiments, the affinity modified IgSF domain is capable ofspecifically binding to the two mammalian cells forming the IS.

In some embodiments according to any one of the immunomodulatoryproteins described above, the wild-type IgSF domain is from an IgSFfamily member of a family selected from Signal-Regulatory Protein (SIRP)Family, Triggering Receptor Expressed On Myeloid Cells Like (TREML)Family, Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM)Family, Sialic Acid Binding Ig-Like Lectin (SIGLEC) Family, ButyrophilinFamily, B7 family, CD28 family, V-set and Immunoglobulin DomainContaining (VSIG) family, V-set transmembrane Domain (VSTM) family,Major Histocompatibility Complex (MHC) family, Signaling lymphocyticactivation molecule (SLAM) family, Leukocyte immunoglobulin-likereceptor (LIR), Nectin (Nec) family, Nectin-like (NECL) family,Poliovirus receptor related (PVR) family, Natural cytotoxicitytriggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM)family or Killer-cell immunoglobulin-like receptors (KIR) family. Insome embodiments, the wild-type IgSF domain is from an IgSF memberselected from CD80, CD86, PD-L1, PD-L2, ICOS Ligand, B7-H3, B7-H4, CD28,CTLA4, PD-1, ICOS, BTLA, CD4, CD8-alpha, CD8-beta, LAG3, TIM-3, CEACAM1,TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R or Nkp30. In someembodiments, the wild-type IgSF domain is a human IgSF member.

In some embodiments according to any one of the immunomodulatoryproteins described above, the wild-type IgSF domain is an IgV domain, anIgC1 domain, an IgC2 domain or a specific binding fragment thereof. Insome embodiments, the affinity-modified IgSF domain is an affinitymodified IgV domain, affinity modified IgC1 domain or an affinitymodified IgC2 domain or is a specific binding fragment thereofcomprising the one or more amino acid substitutions.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein comprises atleast two non-immunoglobulin affinity modified IgSF domains. In someembodiments each of the non-immunoglobulin affinity modified IgSFdomains binds to a different cognate binding partner, wherein the twoaffinity modified IgSF domain specifically binds non-competitively tothe at least two different cognate binding partners. In someembodiments, the at least two non-immunoglobulin affinity modified IgSFdomains each comprise one or more different amino acid substitutions inthe same wild-type IgSF domain. In some embodiments, the at least twonon-immunoglobulin affinity modified IgSF domains each comprise one ormore amino acid substitutions in different wild-type IgSF domains. Insome embodiments, the different wild-type IgSF domains are fromdifferent IgSF family members.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein comprises onlyone non-immunoglobulin affinity modified IgSF domain.

In some embodiments according to any one of the immunomodulatoryproteins described above, the affinity-modified IgSF comprises at least85% sequence identity to a wild-type IgSF domain or a specific bindingfragment thereof contained in the sequence of amino acids set forth inany of SEQ ID NOS: 1-27. In some embodiments, the immunomodulatoryprotein further comprises a second affinity-modified IgSF, wherein thesecond affinity-modified IgSF domain comprises at least 85% sequenceidentity to a wild-type IgSF domain or a specific binding fragmentthereof contained in the sequence of amino acids set forth in any of SEQID NOS: 1-27.

In some embodiments according to any one of the immunomodulatoryproteins described above, the wild-type IgSF domain is a member of theB7 family. In some embodiments, the wild-type IgSF domain is a domain ofCD80, CD86 or ICOSLG. In some embodiments, the wild-type IgSF domain isa domain of CD80.

In some embodiments according to any one of the immunomodulatoryproteins described above, provided herein is an immunomodulatoryprotein, comprising at least one affinity modified CD80 immunoglobulinsuperfamily (IgSF) domain comprising one or more amino acidsubstitution(s) in a wild-type CD80 IgSF domain, wherein the at leastone affinity modified CD80 IgSF domain has increased binding to at leasttwo cognate binding partners compared to the wild-type CD80 IgSF domain.

In some embodiments according to any one of the immunomodulatoryproteins described above, the cognate binding partners are CD28 andPD-L1. In some embodiments, the wild-type IgSF domain is an IgV domainand/or the affinity modified CD80 domain is an affinity modified IgVdomain. In some embodiments, the affinity-modified domain comprises atleast 85% sequence identity to a wild-type CD80 domain or a specificbinding fragment thereof contained in the sequence of amino acids setforth in SEQ ID NO:1.

In some embodiments according to any one of the immunomodulatoryproteins described above, the at least one affinity modified IgSF domaincomprises at least 1 and no more than twenty amino acid substitutions inthe wild-type IgSF domain. In some embodiments, the at least oneaffinity modified IgSF domain comprises at least 1 and no more than tenamino acid substitutions in the wild-type IgSF domain. In someembodiments, the at least one affinity modified IgSF domain comprises atleast 1 and no more than five amino acid substitutions in the wild-typeIgSF domain.

In some embodiments according to any one of the immunomodulatoryproteins described above, the affinity modified IgSF domain has at least120% of the binding affinity as its wild-type IgSF domain to each of theat least two cognate binding partners.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein further comprisesa non-affinity modified IgSF domain.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is soluble. Insome embodiments, the immunomodulatory protein lacks a transmembranedomain or a cytoplasmic domain. In some embodiments, theimmunomodulatory protein comprises only the extracellular domain (ECD)or a specific binding fragment thereof comprising the affinity modifiedIgSF domain.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is glycosylatedor pegylated.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is linked to amultimerization domain. In some embodiments, the immunomodulatoryprotein is linked to an Fc domain or a variant thereof with reducedeffector function. In some embodiments, the Fc domain is an IgG1 domain,an IgG2 domain or is a variant thereof with reduced effector function.In some embodiments, the Fc domain is mammalian, optionally human; orthe variant Fc domain comprises one or more amino acid modificationscompared to an unmodified Fc domain that is mammalian, optionally human.In some embodiments, the Fc domain or variant thereof comprises thesequence of amino acids set forth in SEQ ID NO:226 or SEQ ID NO:227 or asequence of amino acids that exhibits at least 85% sequence identity toSEQ ID NO:226 or SEQ ID NO:227.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is linkedindirectly via a linker.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is a dimer.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is attached to aliposomal membrane.

Also provided are immunomodulatory proteins comprising at least twonon-immunoglobulin immunoglobulin superfamily (IgSF) domains, wherein:at least one is an affinity modified IgSF domain and the at least twonon-immunoglobulin IgSF domains each independently bind to at least onedifferent binding partner. In some embodiments, the at least twonon-immunoglobulin IgSF domains bind non-competitively to the differentbinding partners. In some embodiments, the affinity modified IgSF domaincontains one more amino acid substitutions in a first wild-type orunmodified IgSF domain. In some embodiments, the other of the IgSFdomain is a wild-type or unmodified IgSF domain. In some embodiments,the other of the IgSF domain is also an affinity modified IgSF domain.

In some embodiments according to any of the above immunomodulatoryproteins, the immunomodulatory protein contains at least two affinitymodified non-immunoglobulin IgSF domains (a first affinity modified IgSFdomain and a second affinity modified IgSF domain) in which the firstnon-immunoglobulin modified IgSF domain comprises one or more amino acidsubstitutions in a first wild-type-type IgSF domain and the secondnon-immunoglobulin modified IgSF domain comprises one or more amino acidsubstitutions in a second wild-type IgSF domain, and in which the firstand second non-immunoglobulin modified IgSF domain each specificallybind to at least one different cognate binding partner. In someembodiments, the at least two non-immunoglobulin IgSF domains bindnon-competitively to the different binding partners. In someembodiments, the first and second non-immunoglobulin modified IgSFdomain is affinity-modified to exhibit altered binding to its cognatebinding partner. Thus, in some embodiments, the first and secondnon-immunoglobulin modified IgSF domains are affinity-modified IgSFdomains. In some embodiments, the first non-immunoglobulin modified IgSFdomain exhibits altered binding to at least one of its cognate bindingpartner(s) compared to the first wild-type IgSF domain; and the secondnon-immunoglobulin modified IgSF domain exhibits altered binding to atleast one of its cognate binding partner(s) compared to the secondwild-type IgSF domain. In some embodiments, the altered binding isindependently either increased or decreased.

In some embodiments, the different cognate binding partners are cellsurface molecular species expressed on the surface of a mammalian cell.In some embodiments, the different cell surface molecular species areexpressed in cis configuration or trans configuration. In someembodiments, the mammalian cell is one of two mammalian cells forming animmunological synapse (IS) and the different cell surface molecularspecies is expressed on at least one of the two mammalian cells formingthe IS. In some embodiments, at least one of the mammalian cells is alymphocyte. In some embodiments, the lymphocyte is an NK cell or a Tcell. In some embodiments, binding of the immunomodulatory protein tothe cell modulates the immunological activity of the lymphocyte. In someembodiments, the immunomodulatory protein is capable of effectingincreased immunological activity compared to the wild-type proteincomprising the wild-type IgSF domain. In some embodiments, theimmunomodulatory protein is capable of effecting decreased immunologicalactivity compared to the wild-type protein comprising the wild-type IgSFdomain. In some embodiments, at least one of the mammalian cells is atumor cell. In some embodiments, the mammalian cells are human cells. Insome embodiments, the immunomodulatory protein is capable ofspecifically binding to the two mammalian cells forming the IS.

In some embodiments according to any one of the immunomodulatoryproteins described above, the first and second modified IgSF domainseach comprise one or more amino acid substitutions in differentwild-type IgSF domains. In some embodiments, the different wild-typeIgSF domains are from different IgSF family members. In someembodiments, the first and second modified IgSF domains arenon-wild-type combinations. In some embodiments, the first wild-typeIgSF domain and second wild-type IgSF domain each individually is froman IgSF family member of a family selected from Signal-RegulatoryProtein (SIRP) Family, Triggering Receptor Expressed On Myeloid CellsLike (TREML) Family, Carcinoembryonic Antigen-related Cell AdhesionMolecule (CEACAM) Family, Sialic Acid Binding Ig-Like Lectin (SIGLEC)Family, Butyrophilin Family, B7 family, CD28 family, V-set andImmunoglobulin Domain Containing (VSIG) family, V-set transmembraneDomain (VSTM) family, Major Histocompatibility Complex (MHC) family,Signaling lymphocytic activation molecule (SLAM) family, Leukocyteimmunoglobulin-like receptor (LIR), Nectin (Nec) family, Nectin-like(NECL) family, Poliovirus receptor related (PVR) family, Naturalcytotoxicity triggering receptor (NCR) family, T cell immunoglobulin andmucin (TIM) family or Killer-cell immunoglobulin-like receptors (KIR)family. In some embodiments, the first wild-type IgSF domain and secondwild-type IgSF domain each individually is from an IgSF member selectedfrom CD80, CD86, PD-L1, PD-L2, ICOS Ligand, B7-H3, B7-H4, CD28, CTLA4,PD-1, ICOS, BTLA, CD4, CD8-alpha, CD8-beta, LAG3, TIM-3, CEACAM1, TIGIT,PVR, PVRL2, CD226, CD2, CD160, CD200, CD200R or Nkp30.

In some embodiments according to any one of the immunomodulatoryproteins described above, the first modified IgSF domain and the secondmodified IgSF domain each individually comprises at least 85% sequenceidentity to a wild-type IgSF domain or a specific binding fragmentthereof contained in the sequence of amino acids set forth in any of SEQID NOS: 1-27.

In some embodiments according to any one of the immunomodulatoryproteins described above, the first and second wild-type IgSF domaineach individually is a member of the B7 family. In some embodiments, thefirst and second wild-type IgSF domain each individually is from CD80,CD86 or ICOSLG. In some embodiments, the first or second wild-type IgSFdomain is from a member of the B7 family and the other of the first orsecond wild-type IgSF domain is from another IgSF family.

In some embodiments according to any one of the immunomodulatoryproteins described above, the first and second wild-type IgSF domain isfrom ICOSLG and NKp30.

In some embodiments according to any one of the immunomodulatoryproteins described above, the first and second wild-type IgSF domain isfrom CD80 and NKp30.

In some embodiments according to any one of the immunomodulatoryproteins described above, the first and second wild-type IgSF domaineach individually is a human IgSF member.

In some embodiments according to any one of the immunomodulatoryproteins described above, the first and second wild-type IgSF domaineach individually is an IgV domain, and IgC1 domain, an IgC2 domain or aspecific binding thereof. In some embodiments, the firstnon-immunoglobulin modified domain and the second non-immunoglobulinmodified domain each individually is a modified IgV domain, modifiedIgC1 domain or a modified IgC2 domain or is a specific binding fragmentthereof comprising the one or more amino acid substitutions. In someembodiments, at least one of the first non-immunoglobulin modifieddomain or the second non-immunoglobulin modified domain is a modifiedIgV domain. In some embodiments, the first non-immunoglobulin modifiedIgSF domain and the second non-immunoglobulin modified IgSF domain eachindividually comprise 1 and no more than twenty amino acidsubstitutions. In some embodiments, the first non-immunoglobulinmodified IgSF domain and the second non-immunoglobulin modified IgSFdomain each individually comprise 1 and no more than ten amino acidsubstitutions. In some embodiments, the first non-immunoglobulinmodified IgSF domain and the second non-immunoglobulin modified IgSFdomain each individually comprise 1 and no more than five amino acidsubstitutions.

In some embodiments according to any one of the immunomodulatoryproteins described above, at least one of the first or secondnon-immunoglobulin modified IgSF domain has between 10% and 90% of thebinding affinity of the wild-type IgSF domain to at least one of itscognate binding partner. In some embodiments, at least one of the firstof second non-immunoglobulin modified IgSF domain has at least 120% ofthe binding affinity of the wild-type IgSF domain to at least one of itscognate binding partner.

In some embodiments according to any one of the immunomodulatoryproteins described above, the first and second non-immunoglobulinmodified IgSF domain each individually has at least 120% of the bindingaffinity of the wild-type IgSF domain to at least one of its cognatebinding partner.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is soluble.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is glycosylatedor pegylated.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is linked to amultimerization domain. In some embodiments, the immunomodulatoryprotein is linked to an Fc domain or a variant thereof with reducedeffector function. In some embodiments, the Fc domain is an IgG1 domain,an IgG2 domain or is a variant thereof with reduced effector function.In some embodiments, the Fc domain is mammalian, optionally human; orthe variant Fc domain comprises one or more amino acid modificationscompared to an unmodified Fc domain that is mammalian, optionally human.In some embodiments, the Fc domain or variant thereof comprises thesequence of amino acids set forth in SEQ ID NO:226 or SEQ ID NO:227 or asequence of amino acids that exhibits at least 85% sequence identity toSEQ ID NO:226 or SEQ ID NO:227.

In some embodiments according to any one of the immunomodulatoryproteins described above, the variant CD80 polypeptide is linkedindirectly via a linker.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is a dimer.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein further comprisesone or more additional non-immunoglobulin IgSF domain that is the sameof different from the first or second non-immunoglobulin modified IgSFdomain. In some embodiments, the one or more additionalnon-immunoglobulin IgSF domain is an affinity modified IgSF domain.

In some embodiments according to any one of the immunomodulatoryproteins described above, the immunomodulatory protein is attached to aliposomal membrane.

In some embodiments, provided herein is a nucleic acid molecule,encoding the immunomodulatory polypeptide according to any one of theembodiments described above. In some embodiments, the nucleic acidmolecule is synthetic nucleic acid. In some embodiments, the nucleicacid molecule is cDNA.

In some embodiments, provided herein is a vector, comprising the nucleicacid molecule according to any one of the embodiments described above.In some embodiments, the vector is an expression vector.

In some embodiments, provided herein is a cell, comprising the vector ofaccording to any one of the embodiments described above. In someembodiments, the cell is a eukaryotic cell or prokaryotic cell.

In some embodiments, provided herein is a method of producing animmunomodulatory protein, comprising introducing the nucleic acidmolecule according to any one of the embodiments described above orvector according to any one of the embodiments described above into ahost cell under conditions to express the protein in the cell. In someembodiments, the method further comprises isolating or purifying theimmunomodulatory protein from the cell.

In some embodiments, provided herein is a pharmaceutical composition,comprising the immunomodulatory protein according to any one of theembodiments described above. In some embodiments, the pharmaceuticalcomposition comprises a pharmaceutically acceptable excipient. In someembodiments, the pharmaceutical composition is sterile.

In some embodiments, provided herein is an article of manufacturecomprising the pharmaceutical composition according to any one of theembodiments described above in a vial. In some embodiments, the vial issealed.

In some embodiments, provided herein is a kit comprising thepharmaceutical composition according to any one of the embodimentsdescribed above, and instructions for use.

In some embodiments, provided herein is a kit comprising the article ofmanufacture according to any one of the embodiments described above, andinstructions for use.

In some embodiments, provided herein is a method of modulating an immuneresponse in a subject, comprising administering therapeuticallyeffective amount of the immunomodulatory protein according to any one ofthe embodiments described above to the subject. In some embodiments,modulating the immune response treats a disease or condition in thesubject. In some embodiments, the immune response is increased. In someembodiments, the disease or condition is a tumor or cancer. In someembodiments, the disease or condition is selected from melanoma, lungcancer, bladder cancer or a hematological malignancy. In someembodiments, the immune response is decreased. In some embodiments, thedisease or condition is an inflammatory disease or condition. In someembodiments, the disease or condition is selected from Crohn's disease,ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, orpsoriasis.

In some embodiments, provided herein is a method of identifying anaffinity modified immunomodulatory protein, comprising: a) contacting amodified protein comprising at least one non-immunoglobulin modifiedimmunoglobulin superfamily (IgSF) domain or specific binding fragmentthereof with at least two cognate binding partners under conditionscapable of effecting binding of the protein with the at least twocognate binding partners, wherein the at least one modified IgSF domaincomprises one or more amino acid substitutions in a wild-type IgSFdomain; b) identifying a modified protein comprising the modified IgSFdomain that has increased binding to at least one of the two cognatebinding partners compared to a protein comprising the wild-type IgSFdomain; and c) selecting a modified protein comprising the modified IgSFdomain that binds non-competitively to the at least two cognate bindingpartners, thereby identifying the affinity modified immunomodulatoryprotein. In some embodiments, step b) comprises identifying a modifiedprotein comprising a modified IgSF domain that has increased binding toeach of the at least two cognate binding partners compared to a proteincomprising the wild-type domain. In some embodiments, prior to step a),introducing one or more amino acid substitutions into the wild-type IgSFdomain, thereby generating a modified protein comprising the modifiedIgSF domain. In some embodiments, the modified protein comprises atleast two modified IgSF domains or specific binding fragments thereof,wherein the first IgSF domain comprises one or more amino acidsubstitutions in a first wild-type-type IgSF domain and the secondnon-immunoglobulin affinity modified IgSF domain comprises one or moreamino acid substitutions in a second wild-type IgSF domain. In someembodiments, the first and second non-immunoglobulin affinity modifiedIgSF domain each specifically bind to at least one different cognatebinding partner.

In some embodiments, also provided are immunomodulatory proteincomprising at least one non-immunoglobulin affinity modifiedimmunoglobulin superfamily (IgSF) domain. In some embodiments, theaffinity modified IgSF domain specifically binds non-competitively to atleast two cell-surface molecular species. In some embodiments, each ofthe molecular species is expressed on at least one of two mammaliancells forming an immunological synapse (IS). In some embodiments, themolecular species are in cis configuration or trans configuration. Insome embodiments, one of the mammalian cells is a lymphocyte and bindingof the affinity modified IgSF domain modulates immunological activity ofthe lymphocyte. In some embodiments, the affinity modified IgSF domainspecifically binds to the two mammalian cells forming the IS.

In some embodiments, the immunomodulatory protein comprises at least twonon-immunoglobulin affinity modified IgSF domains and theimmunomodulatory protein specifically binds to the two mammalian cellsforming the IS. In some embodiments, the immunomodulatory proteincomprises at least two affinity modified IgSF domains, wherein theaffinity modified IgSF domains are not the same species of IgSF domain.

In some embodiments, one of the two mammalian cells is a tumor cell. Insome embodiments, the lymphocyte is an NK cell or a T-cell. In someembodiments, the mammalian cells are a mouse, rat, cynomolgus monkey, orhuman cells.

In some embodiments, the IgSF cell surface molecular species are humanIgSF members.

In some embodiments, the immunomodulatory protein comprises an affinitymodified mammalian IgSF member. In some embodiments, the affinitymodified IgSF domain is an affinity modified IgV, IgC1, or IgC2 domain.In some embodiments, the affinity modified IgSF domain differs by atleast one and no more than ten amino acid substitutions from itswild-type IgSF domain. In some embodiments, the affinity modified IgSFdomain differs by at least one and no more than five amino acidsubstitutions from its wild-type IgSF domain.

In some embodiments, the affinity modified human IgSF member is at leastone of: CD80, PVR, ICOSLG, or HAVCR2. In some embodiments, the affinitymodified IgSF domain comprises at least one affinity modified human CD80domain. In some embodiments, the affinity modified IgSF domain is ahuman CD80 IgSF domain.

In some embodiments, the immunomodulatory protein has at least 85%sequence identity with an amino acid sequence selected from SEQ ID NOS:1-27, or a fragment thereof. In some embodiments, the immunomodulatoryprotein has at least 90% sequence identity with an amino acid sequenceselected from SEQ ID NOS: 1-27 or a fragment thereof. In someembodiments, the immunomodulatory protein has at least 95% sequenceidentity with an amino acid sequence selected from SEQ ID NOS: 1-27, ora fragment thereof. In some embodiments, the immunomodulatory proteinhas at least 99% sequence identity with an amino acid sequence selectedfrom SEQ ID NOS: 1-27, or a fragment thereof. In some embodiments, theimmunomodulatory protein having at least 85%, 90%, 95%, or 99% sequenceidentity with an amino acid sequence selected from SEQ ID NOS: 1-27 or afragment thereof further comprises a second immunomodulatory protein,wherein the second immunomodulatory protein has at least 85%, 90%, 95%,or 99% sequence identity with an amino acid sequence selected from SEQID NOS: 1-27 or a fragment thereof.

In some embodiments, the immunological activity is enhanced. In othersit's suppressed.

In some embodiments, the affinity modified IgSF domain has between 10%and 90% of the binding affinity of the wild-type IgSF domain to at leastone of the two cell surface molecular species. In some embodiments, theaffinity modified IgSF domain specifically binds non-competitively toexactly one IgSF member. In some embodiments, the affinity modified IgSFdomain has at least 120% of the binding affinity as its wild-type IgSFdomain to at least one of the two cell surface molecular species.

In some embodiments, the immunomodulatory protein is covalently bonded,directly or indirectly, to an antibody fragment crystallizable (Fc). Insome embodiments, the immunomodulatory protein is glycosylated orpegylated. In some embodiments, the immunomodulatory protein is soluble.In some embodiments, the immunomodulatory protein is embedded in aliposomal membrane. In some embodiments, the immunomodulatory protein isdimerized by intermolecular disulfide bonds.

In some embodiments, the immunomodulatory protein is in apharmaceutically acceptable carrier.

In another aspect, provided herein are immunomodulatory proteincomprising at least two non-immunoglobulin affinity modifiedimmunoglobulin superfamily (IgSF) domains. The affinity modified IgSFdomains each specifically binds to its own cell surface molecularspecies. Each of the molecular species is expressed on at least one ofthe two mammalian cells forming an immunological synapse (IS) and one ofthe mammalian cells is a lymphocyte. The molecular species are, in someembodiments, in cis configuration or trans configuration. Binding of theaffinity modified IgSF domain modulates immunological activity of thelymphocyte. In some embodiments, at least one of the affinity modifiedIgSF domains binds competitively. In some embodiments, the affinitymodified IgSF domains are not the same species of IgSF domain. In someembodiments, the affinity modified IgSF domains are non-wild typecombinations. In some embodiments, the cell surface molecular speciesare human IgSF members. In some embodiments, the at least two affinitymodified IgSF domains are from at least one of: CD80, CD86, CD274,PDCD1LG2, ICOSLG, CD276, VTCN1, CD28, CTLA4, PDCD1, ICOS, BTLA, CD4,CD8A, CD8B, LAG3, HAVCR2, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160,CD200, NKp30, or CD200R1.

In some embodiments, the immunomodulatory protein comprises at least twoaffinity modified mammalian IgSF members. In some embodiments, themammalian IgSF members are human IgSF members. In some embodiments, themammalian IgSF members are at least two of: CD80, CD86, CD274, PDCD1LG2,ICOSLG, CD276, VTCN1, CD28, CTLA4, PDCD1, ICOS, BTLA, CD4, CD8A, CD8B,LAG3, HAVCR2, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200,NKp30, or CD200R1. In some embodiments, immunological activity isenhanced. In some embodiments, immunological activity is suppressed. Insome embodiments, one of the two mammalian cells is a tumor cell. Insome embodiments, the lymphocyte is an NK cell or a T-cell. In someembodiments, the mammalian cells are a mouse, rat, cynomolgus monkey, orhuman cells. In some embodiments, at least one of the two affinitymodified IgSF domains has between 10% and 90% of the binding affinity ofthe wild-type IgSF domain to at least one of the cell surface molecularspecies. In some embodiments, at least one of the two affinity modifiedIgSF domains specifically binds to exactly one cell surface molecularspecies. In some embodiments, at least one of the two affinity modifiedIgSF domains has at least 120% of the binding affinity as its wild-typeIgSF domain to at least one of the two cell surface molecular species.In some embodiments, the affinity modified IgSF domains are at least oneof an IgV, IgC1, or IgC2 domain. In some embodiments, each of the atleast two affinity modified IgSF domains differs by at least one and nomore than ten amino acid substitutions from its wild-type IgSF domain.In some embodiments, each of the at least two affinity modified IgSFdomains differs by at least one and no more than five amino acidsubstitutions from its wild-type IgSF domain. In some embodiments, theimmunomodulatory protein is covalently bonded, directly or indirectly,to an antibody fragment crystallizable (Fc). In some embodiments, theimmunomodulatory protein is in a pharmaceutically acceptable carrier. Insome embodiments, the immunomodulatory protein is glycosylated orpegylated. In some embodiments, the immunomodulatory protein is soluble.In some embodiments, the immunomodulatory protein is embedded in aliposomal membrane. In some embodiments, the immunomodulatory protein isdimerized by intermolecular disulfide bonds.

In another aspect, the present invention relates to a recombinantnucleic acid encoding any of the immunomodulatory proteins summarizedabove.

In another aspect, the present invention relates to a recombinantexpression vector comprising a nucleic acid encoding any of theimmunomodulatory proteins summarized above.

In another aspect, the present invention relates to a recombinant hostcell comprising an expression vector as summarized above.

In another aspect, the present invention relates to a method of makingany of the immunomodulatory proteins summarized above. The methodcomprises culturing the recombinant host cell under immunomodulatoryprotein expressing conditions, expressing the immunomodulatory proteinencoded by the recombinant expression vector therein, and purifying therecombinant immunomodulatory protein expressed thereby.

In another aspect, the present invention relates to a method of treatinga mammalian patient in need of an enhanced or suppressed immunologicalresponse by administering a therapeutically effective amount of animmunomodulatory protein of any of the embodiments described above. Insome embodiments, the enhanced immunological response treats melanoma,lung cancer, bladder cancer, or a hematological malignancy in thepatient. In some embodiments, the suppressed immunological responsetreats Crohn's disease, ulcerative colitis, multiple sclerosis, asthma,rheumatoid arthritis, or psoriasis in the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts results of a competition binding assay for binding ofbiotinylated recombinant CD28 Fc fusion protein (rCD28.Fc) toimmobilized CD80 variant A91G ECD-Fc fusion molecule in the presence ofunlabeled recombinant human PD-L1-his, human CTLA-4-his orhuman-PD-L2-Fc fusion protein.

FIG. 1B depicts results of a competition binding assay for binding ofbiotinulated recombinant human PD-L1-his monomeric protein toimmobilized CD80 variant A91G ECD-Fc fusion molecule in the presence ofunlabeled recombinant human rCD28.Fc, human CTLA-4.Fc or human PD-L2.Fc

INCORPORATION BY REFERENCE

All publications, including patents, patent applications scientificarticles and databases, mentioned in this specification are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, including patent, patentapplication, scientific article or database, were specifically andindividually indicated to be incorporated by reference. If a definitionset forth herein is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

DETAILED DESCRIPTION

Provided herein are soluble immunomodulatory proteins that are capableof binding to one or more protein ligands, and generally two or moreprotein ligands, to modulate, e.g. induce, enhance or suppress,immunological immune responses. In some embodiments, the protein ligandsare cell surface proteins expressed by immune cells that engage with oneor more other immune receptor, e.g. on lymphocytes, to induce inhibitoryor activating signals. For example, the interaction of certain receptorson lymphocytes with their cognate cell surface ligands to form animmunological synapse (IS) between antigen-presenting cells (APCs) ortarget cells and lymphocytes can provide costimulatory or inhibitorysignals that can regulate the immune system. In some aspects, theimmunomodulatory proteins provided herein can alter the interaction ofcell surface protein ligands with their receptors to thereby modulateimmune cells, such as T cell, activity.

In some embodiments, under normal physiological conditions, the Tcell-mediated immune response is initiated by antigen recognition by theT cell receptor (TCR) and is regulated by a balance of co-stimulatoryand inhibitory signals (i.e., immune checkpoint proteins). The immunesystem relies on immune checkpoints to prevent autoimmunity (i.e.,self-tolerance) and to protect tissues from excessive damage during animmune response, for example during an attack against a pathogenicinfection. In some cases, however, these immunomodulatory proteins canbe dysregulated in diseases and conditions, including tumors, as amechanism for evading the immune system.

Thus, in some aspects, immunotherapy that alters immune cell activity,such as T cell activity, can treat certain diseases and conditions inwhich the immune response is dysregulated. Therapeutic approaches thatseek to modulate interactions in the IS would benefit from the abilityto bind multiple IS targets simultaneously and in a manner that issensitive to temporal sequence and spatial orientation. Currenttherapeutic approaches fall short of this goal. Instead, solublereceptors and antibodies typically bind no more than a single targetprotein at a time. This may be due to the absence of more than a singletarget species. Additionally, wild-type receptors and ligands possesslow affinities for cognate binding partners, which preclude their use assoluble therapeutics.

Less trivially, however, soluble receptors and antibodies generally bindcompetitively (e.g., to no more than one target species at a time) andtherefore lack the ability to simultaneously bind multiple targets. Andwhile bispecific antibodies, as well as modalities comprising dualantigen binding regions, can bind to more than one target moleculesimultaneously, the three-dimensional configuration typical of thesemodalities often precludes them intervening in key processes occurringin the IS in a manner consistent with their temporal and spatialrequirements.

What is needed is an entirely new class of therapeutic molecules thathave the specificity and affinity of antibodies or soluble receptorsbut, in addition, maintain the size, volume, and spatial orientationconstraints required in the IS and possess increased affinity towardsrespective cognate binding partners. Further, such therapeutics wouldhave the ability to bind to their targets non-competitively as well ascompetitively. A molecule with these properties would therefore havenovel function in the ability to integrate into multi-protein complexesat IS and generate the desired binding configuration and resultingbiological activity.

To this end, emerging immuno-oncology therapeutic regimes need to safelybreak tumor-induced T cell tolerance. Current state-of-the-artimmuno-therapeutics block PD-1 or CTLA4, central inhibitory molecules ofthe B7/CD28 family that are known to limit T cell effector function.While antagonistic antibodies against such single targets function todisrupt immune synapse checkpoint signaling complexes, they fall shortof simultaneously activating T cells. Conversely, bispecific antibodyapproaches activate T cells, but fall short of simultaneously blockinginhibitory ligands that regulate the induced signal.

To address these shortcomings, provided are therapeutic molecules that,in some embodiments, will both stimulate T-cell activation signaling andblock inhibitory regulation simultaneously. In some embodiments, theprovided immunomodulatory proteins relate to immunoglobulin superfamily(IgSF) components of the immune synapse that are known to have a dualrole in both T-cell activation and blocking of inhibitory ligands. Inparticular aspects, the provided immunomodulatory proteins provide animmunotherapy platform using affinity modified native immune ligands togenerate immunotherapy biologics that bind with tunable affinities toone or more of their cognate immune receptors in the treatment of avariety of oncological and immunological indications. In some aspects,IgSF based therapeutics engineered from immune system ligands, such ashuman immune system ligands, themselves are more likely to retain theirability to normally assemble into key pathways of the immune synapse andmaintain normal interactions and regulatory functions in ways thatantibodies or next-generation bi-specific reagents cannot. This is dueto the relatively large size of antibodies as well as from the fact theyare not natural components of the immune synapse. These unique featuresof human immune system ligands promise to provide a new level ofimmunotherapeutic efficacy and safety.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. Definitions

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

The terms used throughout this specification are defined as followsunless otherwise limited in specific instances. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific terms,acronyms, and abbreviations used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains. Unless indicated otherwise, abbreviations andsymbols for chemical and biochemical names is per IUPAC-IUBnomenclature. Unless indicated otherwise, all numerical ranges areinclusive of the values defining the range as well as all integer valuesin-between.

The term “affinity modified” as used in the context of an immunoglobulinsuperfamily domain, means a mammalian immunoglobulin superfamily (IgSF)domain having an altered amino acid sequence (relative to the wild-typecontrol IgSF domain) such that it has an increased or decreased bindingaffinity or avidity to at least one of its cognate binding partnerscompared to the wild-type (i.e., non-affinity modified) IgSF controldomain. In some embodiments, the IgSF domain can contain 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more amino acid differences, such as aminoacid substitutions, in a wildtype or unmodified IgSF domain. An IgSFdomain having an altered amino acid sequence (relative to the wild-typecontrol IgSF domain) which does not have an increased or decreasedbinding affinity or avidity to at least one of its cognate bindingpartners compared to the wild-type IgSF control domain is a non-affinitymodified IgSF domain. An increase or decrease in binding affinity oravidity can be determined using well known binding assays such as flowcytometry. Larsen et al., American Journal of Transplantation, Vol 5:443-453 (2005). See also, Linsley et al., Immunity, 1: 7930801 (1994).An increase in a protein's binding affinity or avidity to its cognatebinding partner(s) is to a value at least 10% greater than that of thewild-type IgSF domain control and in some embodiments, at least 20%,30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greaterthan that of the wild-type IgSF domain control value. A decrease in aprotein's binding affinity or avidity to at least one of its cognatebinding partner is to a value no greater than 90% of the control but noless than 10% of the wild-type IgSF domain control value, and in someembodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, or 20% but noless than 10% of the wild-type IgSF domain control value. An affinitymodified protein is altered in primary amino acid sequence bysubstitution, addition, or deletion of amino acid residues. The term“affinity modified IgSF domain” is not be construed as imposing anycondition for any particular starting composition or method by which theaffinity modified IgSF domain was created. Thus, the affinity modifiedIgSF domains of the present invention are not limited to wild type IgSFdomains that are then transformed to an affinity modified IgSF domain byany particular process of affinity modification. An affinity modifiedIgSF domain polypeptide can, for example, be generated starting fromwild type mammalian IgSF domain sequence information, then modeled insilico for binding to its cognate binding partner, and finallyrecombinantly or chemically synthesized to yield the affinity modifiedIgSF domain composition of matter. In but one alternative example, anaffinity modified IgSF domain can be created by site-directedmutagenesis of a wild-type IgSF domain. Thus, affinity modified IgSFdomain denotes a product and not necessarily a product produced by anygiven process. A variety of techniques including recombinant methods,chemical synthesis, or combinations thereof, may be employed.

The terms “binding affinity,” and “binding avidity” as used herein meansthe specific binding affinity and specific binding avidity,respectively, of a protein for its cognate binding partner underspecific binding conditions. In biochemical kinetics avidity refers tothe accumulated strength of multiple affinities of individualnon-covalent binding interactions, such as between an IgSF domain andits cognate binding partner. As such, avidity is distinct from affinity,which describes the strength of a single interaction. Methods fordetermining binding affinity or avidity are known in art. See, forexample, Larsen et al., American Journal of Transplantation, Vol 5:443-453 (2005).

The term “biological half-life” refers to the amount of time it takesfor a substance, such as an immunomodulatory polypeptide of the presentinvention, to lose half of its pharmacologic or physiologic activity orconcentration. Biological half-life can be affected by elimination,excretion, degradation (e.g., enzymatic) of the substance, or absorptionand concentration in certain organs or tissues of the body. In someembodiments, biological half-life can be assessed by determining thetime it takes for the blood plasma concentration of the substance toreach half its steady state level (“plasma half-life”). Conjugates thatcan be used to derivatize and increase the biological half-life ofpolypeptides of the invention are known in the art and include, but arenot limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES),XTEN (extended recombinant peptides; see, WO2013130683), human serumalbumin (HSA), bovine serum albumin (BSA), lipids (acylation), andpoly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

The term “cognate binding partner,” in reference to a protein, such asan IgSF domain or an affinity modified IgSF domain, refers to at leastone molecule (typically a native mammalian protein) to which thereferenced protein specifically binds under specific binding conditions.A species of ligand recognized and specifically binding to its cognatereceptor under specific binding conditions is an example of a cognatebinding partner of that receptor. A “cognate cell surface bindingpartner” is a cognate binding partner expressed on a mammalian cellsurface. In the present invention a “cell surface molecular species” isa cognate binding partner of the immunological synapse (IS), expressedon and by cells, such as mammalian cells, forming the immunologicalsynapse.

The term “competitive binding” as used herein means that a protein iscapable of specifically binding to at least two cognate binding partnersbut that specific binding of one cognate binding partner inhibits, suchas prevents or precludes, simultaneous binding of the second cognatebinding partner. Thus, in some cases, it is not possible for a proteinto bind the two cognate binding partners at the same time. Generally,competitive binders contain the same or overlapping binding site forspecific binding but this is not a requirement. In some embodiments,competitive binding causes a measurable inhibition (partial or complete)of specific binding of a protein to one of its cognate binding partnerdue to specific binding of a second cognate binding partner. A varietyof methods are known to quantify competitive binding such as ELISA(enzyme linked immunosorbent assay) assays.

The term “conservative amino acid substitution” as used herein means anamino acid substitution in which an amino acid residue is substituted byanother amino acid residue having a side chain R group with similarchemical properties (e.g., charge or hydrophobicity). Examples of groupsof amino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine, andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartic acidand glutamic acid; and 7) sulfur-containing side chains: cysteine andmethionine. Conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

The term, “corresponding to” with reference to positions of a protein,such as recitation that nucleotides or amino acid positions “correspondto” nucleotides or amino acid positions in a disclosed sequence, such asset forth in the Sequence listing, refers to nucleotides or amino acidpositions identified upon alignment with the disclosed sequence based onstructural sequence alignment or using a standard alignment algorithm,such as the GAP algorithm. By aligning the sequences, one skilled in theart can identify corresponding residues, for example, using conservedand identical amino acid residues as guides.

The term “cytokine” includes, e.g., but is not limited to, interleukins,interferons (IFN), chemokines, hematopoietic growth factors, tumornecrosis factors (TNF), and transforming growth factors. In general,these are small molecular weight proteins that regulate maturation,activation, proliferation, and differentiation of cells of the immunesystem.

The terms “decrease” or “attenuate” “or suppress” as used herein meansto decrease by a statistically significant amount. A decrease can be atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

The terms “derivatives” or “derivatized” refer to modification of animmunomodulatory protein by covalently linking it, directly orindirectly, so as to alter such characteristics as half-life,bioavailability, immunogenicity, solubility, toxicity, potency, orefficacy while retaining or enhancing its therapeutic benefit.Derivatives can be made by glycosylation, pegylation, lipidation, orFc-fusion.

As used herein, domain (typically a sequence of three or more, generally5 or 7 or more amino acids, such as 10 to 200 amino acid residues)refers to a portion of a molecule, such as a protein or encoding nucleicacid, that is structurally and/or functionally distinct from otherportions of the molecule and is identifiable. For example, domainsinclude those portions of a polypeptide chain that can form anindependently folded structure within a protein made up of one or morestructural motifs and/or that is recognized by virtue of a functionalactivity, such as binding activity. A protein can have one, or more thanone, distinct domains. For example, a domain can be identified, definedor distinguished by homology of the primary sequence or structure torelated family members, such as homology to motifs. In another example,a domain can be distinguished by its function, such as an ability tointeract with a biomolecule, such as a cognate binding partner. A domainindependently can exhibit a biological function or activity such thatthe domain independently or fused to another molecule can perform anactivity, such as, for example binding. A domain can be a linearsequence of amino acids or a non-linear sequence of amino acids. Manypolypeptides contain a plurality of domains. Such domains are known, andcan be identified by those of skill in the art. For exemplificationherein, definitions are provided, but it is understood that it is wellwithin the skill in the art to recognize particular domains by name. Ifneeded appropriate software can be employed to identify domains. It isunderstood that reference to amino acids, including to a specificsequence set forth as a SEQ ID NO used to describe domain organizationof an IgSF domain are for illustrative purposes and are not meant tolimit the scope of the embodiments provided. It is understood thatpolypeptides and the description of domains thereof are theoreticallyderived based on homology analysis and alignments with similarmolecules. Thus, the exact locus can vary, and is not necessarily thesame for each protein. Hence, the specific IgSF domain, such as specificIgV domain or IgC domain, can be several amino acids (one, two, three orfour) longer or shorter.

The term “ectodomain” as used herein refers to the region of a membraneprotein, such as a transmembrane protein, that lies outside thevesicular membrane. Ectodomains often comprise binding domains thatspecifically bind to ligands or cell surface receptors. The ectodomainof a cellular transmembrane protein is alternately referred to as anextracellular domain.

The terms “effective amount” or “therapeutically effective amount” referto a quantity and/or concentration of a therapeutic composition of theinvention, that when administered ex vivo (by contact with a cell from apatient) or in vivo (by administration into a patient) either alone(i.e., as a monotherapy) or in combination with additional therapeuticagents, yields a statistically significant inhibition of diseaseprogression as, for example, by ameliorating or eliminating symptomsand/or the cause of the disease. An effective amount for treating animmune system disease or disorder may be an amount that relieves,lessens, or alleviates at least one symptom or biological response oreffect associated with the disease or disorder, prevents progression ofthe disease or disorder, or improves physical functioning of thepatient. In some embodiments the patient is a human patient.

The terms “enhanced” or “increased” as used herein in the context ofincreasing immunological activity of a mammalian lymphocyte means toincrease interferon gamma (IFN-gamma) production, such as by astatistically significant amount. In some embodiments, the immunologicalactivity can be assessed in a mixed lymphocyte reaction (MLR) assay.Methods of conducting MLR assays are known in the art. Wang et al.,Cancer Immunol Res. 2014 September: 2(9):846-56. In some embodiments anenhancement can be an increase of at least 10%, 20%, 30%, 40%, 50%, 75%,100%, 200%, 300%, 400%, or 500% greater than a non-zero control value.

The term “host cell” refers to a cell that can be used to express aprotein encoded by a recombinant expression vector. A host cell can be aprokaryote, for example, E. coli, or it can be a eukaryote, for example,a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media or CHO strain DX-B11,which is deficient in DHFR.

The term “immunological synapse” or “immune synapse” as used hereinmeans the interface between a mammalian cell that expresses MHC I (majorhistocompatibility complex) or MHC II, such as an antigen-presentingcell or tumor cell, and a mammalian lymphocyte such as an effector Tcell or Natural Killer (NK) cell.

The term “immunoglobulin” (abbreviated “Ig”) as used herein issynonymous with the term “antibody” (abbreviated “Ab”) and refers to amammalian immunoglobulin protein including any of the five humanclasses: IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG(which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. Theterm is also inclusive of immunoglobulins that are less thanfull-length, whether wholly or partially synthetic (e.g., recombinant orchemical synthesis) or naturally produced, such as antigen bindingfragment (Fab), variable fragment (Fv) containing V_(H) and V_(L), thesingle chain variable fragment (scFv) containing V_(H) and V_(L) linkedtogether in one chain, as well as other antibody V region fragments,such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody, Fc, and Fd polypeptidefragments. Bispecific antibodies, homobispecific and heterobispecific,are included within the meaning of the term.

An Fc (fragment crystallizable) region or domain of an immunoglobulinmolecule (also termed an Fc polypeptide) corresponds largely to theconstant region of the immunoglobulin heavy chain, and is responsiblefor various functions, including the antibody's effector function(s). Animmunoglobulin Fc fusion (“Fc-fusion”) is a molecule comprising one ormore polypeptides (or one or more small molecules) operably linked to anFc region of an immunoglobulin. An Fc-fusion may comprise, for example,the Fc region of an antibody (which facilitates pharmacokinetics) andthe IgSF domain of a wild-type or affinity modified immunoglobulinsuperfamily domain (“IgSF”), or other protein or fragment thereof. Insome embodiments, the Fc additionally facilitates effector functions. Insome embodiments, the Fc is a variant Fc that exhibits reduced (e.g.reduced greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) activityto facilitate an effector function. The IgSF domain mediates recognitionof the cognate binding partner (comparable to that of antibody variableregion of an antibody for an antigen). An immunoglobulin Fc region maybe linked indirectly or directly to one or more polypeptides or smallmolecules (fusion partners). Various linkers are known in the art andcan be used to link an Fc to a fusion partner to generate an Fc-fusion.An Fc-fusion protein of the invention typically comprises animmunoglobulin Fc region covalently linked, directly or indirectly, toat least one affinity modified IgSF domain. Fc-fusions of identicalspecies can be dimerized to form Fc-fusion homodimers, or usingnon-identical species to form Fc-fusion heterodimers.

The term “immunoglobulin superfamily” or “IgSF” as used herein means thegroup of cell surface and soluble proteins that are involved in therecognition, binding, or adhesion processes of cells. Molecules arecategorized as members of this superfamily based on shared structuralfeatures with immunoglobulins (i.e., antibodies); they all possess adomain known as an immunoglobulin domain or fold. Members of the IgSFinclude cell surface antigen receptors, co-receptors and co-stimulatorymolecules of the immune system, molecules involved in antigenpresentation to lymphocytes, cell adhesion molecules, certain cytokinereceptors and intracellular muscle proteins. They are commonlyassociated with roles in the immune system. Proteins in theimmunological synapse are often members of the IgSF. IgSF can also beclassified into “subfamilies” based on shared properties such asfunction. Such subfamilies typically consist of from 4 to 30 IgSFmembers.

The terms “IgSF domain” or “immunoglobulin domain” or “Ig domain” asused herein refers a structural domain of IgSF proteins. Ig domains arenamed after the immunoglobulin molecules. They contain about 70-110amino acids and are categorized according to their size and function.Ig-domains possess a characteristic Ig-fold, which has a sandwich-likestructure formed by two sheets of antiparallel beta strands.Interactions between hydrophobic amino acids on the inner side of thesandwich and highly conserved disulfide bonds formed between cysteineresidues in the B and F strands, stabilize the Ig-fold. One end of theIg domain has a section called the complementarity determining regionthat is important for the specificity of antibodies for their ligands.The Ig like domains can be classified (into classes) as: IgV, IgC1,IgC2, or IgI. Most Ig domains are either variable (IgV) or constant(IgC). IgV domains with 9 beta strands are generally longer than IgCdomains with 7 beta strands. Ig domains of some members of the IgSFresemble IgV domains in the amino acid sequence, yet are similar in sizeto IgC domains. These are called IgC2 domains, while standard IgCdomains are called IgC1 domains. T-cell receptor (TCR) chains containtwo Ig domains in the extracellular portion; one IgV domain at theN-terminus and one IgC1 domain adjacent to the cell membrane.

The term “IgSF species” as used herein means an ensemble of IgSF memberproteins with identical or substantially identical primary amino acidsequence. Each mammalian immunoglobulin superfamily (IgSF) memberdefines a unique identity of all IgSF species that belong to that IgSFmember. Thus, each IgSF family member is unique from other IgSF familymembers and, accordingly, each species of a particular IgSF familymember is unique from the species of another IgSF family member.Nevertheless, variation between molecules that are of the same IgSFspecies may occur owing to differences in post-translationalmodification such as glycosylation, phosphorylation, ubiquitination,nitrosylation, methylation, acetylation, and lipidation. Additionally,minor sequence differences within a single IgSF species owing to genepolymorphisms constitute another form of variation within a single IgSFspecies as do wild type truncated forms of IgSF species owing to, forexample, proteolytic cleavage. A “cell surface IgSF species” is an IgSFspecies expressed on the surface of a cell, generally a mammalian cell.

The term “immunological activity” as used herein in the context ofmammalian lymphocytes means their expression of cytokines, such aschemokines or interleukins. Assays for determining enhancement orsuppression of immunological activity include MLR assays forinterferon-gamma (Wang et al., Cancer Immunol Res. 2014 September:2(9):846-56), SEB (staphylococcal enterotoxin B) T cell stimulationassay (Wang et al., Cancer Immunol Res. 2014 September: 2(9):846-56),and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med.2010: 8: 104). Induction of an immune response results in an increase inimmunological activity relative to quiescent lymphocytes. Animmunomodulatory protein or affinity modified IgSF domain of theinvention can in some embodiments increase or, in alternativeembodiments, decrease IFN-gamma (interferon-gamma) expression in aprimary T-cell assay relative to a wild-type IgSF member or IgSF domaincontrol. Those of skill will recognize that the format of the primaryT-cell assay used to determine an increase in IFN-gamma expression willdiffer from that employed to assay for a decrease in IFN-gammaexpression. In assaying for the ability of an immunomodulatory proteinor affinity modified IgSF domain of the invention to decrease IFN-gammaexpression in a primary T-cell assay, a Mixed Lymphocyte Reaction (MLR)assay can be used as described in Example 6. Conveniently, a solubleform of an affinity modified IgSF domain of the invention can beemployed to determine its ability to antagonize and thereby decrease theIFN-gamma expression in a MLR as likewise described in Example 6.Alternatively, in assaying for the ability of an immunomodulatoryprotein or affinity modified IgSF domain of the invention to increaseIFN-gamma expression in a primary T-cell assay, a co-immobilizationassay can be used. In a co-immobilization assay, a T-cell receptorsignal, provided in some embodiments by anti-CD3 antibody, is used inconjunction with a co-immobilized affinity modified IgSF domain todetermine the ability to increase IFN-gamma expression relative to awild-type IgSF domain control.

An “immunomodulatory protein” is a protein that modulates immunologicalactivity. By “modulation” or “modulating” an immune response is meantthat immunological activity is either enhanced or suppressed. Animmunomodulatory protein can be a single polypeptide chain or a multimer(dimers or higher order multimers) of at least two polypeptide chainscovalently bonded to each other by, for example, interchain disulfidebonds. Thus, monomeric, dimeric, and higher order multimeric proteinsare within the scope of the defined term. Multimeric proteins can behomomultimeric (of identical polypeptide chains) or heteromultimeric (ofdifferent polypeptide chains).

The term “increase” as used herein means to increase by a statisticallysignificant amount. An increase can be at least 5%, 10%, 20%, 30%, 40%,50%, 75%, 100%, or greater than a non-zero control value.

The term “lymphocyte” as used herein means any of three subtypes ofwhite blood cell in a mammalian immune system. They include naturalkiller cells (NK cells) (which function in cell-mediated, cytotoxicinnate immunity), T cells (for cell-mediated, cytotoxic adaptiveimmunity), and B cells (for humoral, antibody-driven adaptive immunity).T cells include: T helper cells, cytotoxic T-cells, natural killerT-cells, memory T-cells, regulatory T-cells, or gamma delta T-cells.Innate lymphoid cells (ILC) are also included within the definition oflymphocyte.

The terms “mammal,” “subject,” or “patient” specifically includesreference to at least one of a: human, chimpanzee, rhesus monkey,cynomolgus monkey, dog, cat, mouse, or rat.

The terms “modulating” or “modulate” as used herein in the contest of animmune response, such as a mammalian immune response, refer to anyalteration, such as an increase or a decrease, of an existing orpotential immune responses that occurs as a result of administration ofan immunomodulatory protein of the present invention. Thus, it refers toan alteration, such as an increase or decrease, of an immune response ascompared to the immune response that occurs or is present in the absenceof the administration of the immunomodulatory protein. Such modulationincludes any induction, or alteration in degree or extent, orsuppression of immunological activity of an immune cell. Immune cellsinclude B cells, T cells, NK (natural killer) cells, NK T cells,professional antigen-presenting cells (APCs), and non-professionalantigen-presenting cells, and inflammatory cells (neutrophils,macrophages, monocytes, eosinophils, and basophils). Modulation includesany change imparted on an existing immune response, a developing immuneresponse, a potential immune response, or the capacity to induce,regulate, influence, or respond to an immune response. Modulationincludes any alteration in the expression and/or function of genes,proteins and/or other molecules in immune cells as part of an immuneresponse. Modulation of an immune response or modulation ofimmunological activity includes, for example, the following:elimination, deletion, or sequestration of immune cells; induction orgeneration of immune cells that can modulate the functional capacity ofother cells such as autoreactive lymphocytes, antigen presenting cells,or inflammatory cells; induction of an unresponsive state in immunecells (i.e., anergy); enhancing or suppressing the activity or functionof immune cells, including but not limited to altering the pattern ofproteins expressed by these cells. Examples include altered productionand/or secretion of certain classes of molecules such as cytokines,chemokines, growth factors, transcription factors, kinases,costimulatory molecules, or other cell surface receptors or anycombination of these modulatory events. Modulation can be assessed, forexample, by an alteration in IFN-gamma (interferon gamma) expressionrelative to or as compared to the wild-type or unmodified IgSF domain(s)control in a primary T cell assay (see, Zhao and Ji, Exp Cell Res. 2016Jan. 1; 340(1) 132-138).

The term “molecular species” as used herein means an ensemble ofproteins with identical or substantially identical primary amino acidsequence. Each mammalian immunoglobulin superfamily (IgSF) memberdefines a collection of identical or substantially identical molecularspecies. Thus, for example, human CD80 is an IgSF member and each humanCD80 molecule is a species of CD80. Variation between molecules that areof the same molecular species may occur owing to differences inpost-translational modification such as glycosylation, phosphorylation,ubiquitination, nitrosylation, methylation, acetylation, and lipidation.Additionally, minor sequence differences within a single molecularspecies owing to gene polymorphisms constitute another form of variationwithin a single molecular species as do wild type truncated forms of asingle molecular species owing to, for example, proteolytic cleavage. A“cell surface molecular species” is a molecular species expressed on thesurface of a mammalian cell. Two or more different species of protein,each of which is present exclusively on one or exclusively the other(but not both) of the two mammalian cells forming the IS, are said to bein “cis” or “cis configuration” with each other. Two different speciesof protein, the first of which is exclusively present on one of the twomammalian cells forming the IS and the second of which is presentexclusively on the second of the two mammalian cells forming the IS, aresaid to be in “trans” or “trans configuration.” Two different species ofprotein each of which is present on both of the two mammalian cellsforming the IS are in both cis and trans configurations on these cells.

The term “non-competitive binding” as used herein means the ability of aprotein to specifically bind simultaneously to at least two cognatebinding partners. In some embodiments, the binding occurs under specificbinding conditions. Thus, the protein is able to bind to at least twodifferent cognate binding partners at the same time, although thebinding interaction need not be for the same duration such that, in somecases, the protein is specifically bound to only one of the cognatebinding partners. In some embodiments, the simultaneous binding is suchthat binding of one cognate binding partner does not substantiallyinhibit simultaneous binding to a second cognate binding partner. Insome embodiments, non-competitive binding means that binding a secondcognate binding partner to its binding site on the protein does notdisplace the binding of a first cognate binding partner to its bindingsite on the protein. Methods of assessing non-competitive binding arewell known in the art such as the method described in Perez de La Lastraet al., Immunology, 1999 April: 96(4): 663-670. In some cases, innon-competitive interactions, the first cognate binding partnerspecifically binds at an interaction site that does not overlap with theinteraction site of the second cognate binding partner such that bindingof the second cognate binding partner does not directly interfere withthe binding of the first cognate binding partner. Thus, any effect onbinding of the cognate binding partner by the binding of the secondcognate binding partner is through a mechanism other than directinterference with the binding of the first cognate binding partner. Forexample, in the context of enzyme-substrate interactions, anon-competitive inhibitor binds to a site other than the active site ofthe enzyme. Non-competitive binding encompasses uncompetitive bindinginteractions in which a second cognate binding partner specificallybinds at an interaction site that does not overlap with the binding ofthe first cognate binding partner but binds to the second interactionsite only when the first interaction site is occupied by the firstcognate binding partner.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyto refer to a polymer of nucleic acid residues (e.g.,deoxyribonucleotides or ribonucleotides) in either single- ordouble-stranded form. Unless specifically limited, the terms encompassnucleic acids containing known analogues of natural nucleotides and thathave similar binding properties to it and are metabolized in a mannersimilar to naturally-occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary nucleotide sequences as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues. The term nucleic acid orpolynucleotide encompasses cDNA or mRNA encoded by a gene.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a mammalian subject, often a human. Apharmaceutical composition typically comprises an effective amount of anactive agent (e.g., an immunomodulatory protein of the invention) and acarrier, excipient, or diluent. The carrier, excipient, or diluent istypically a pharmaceutically acceptable carrier, excipient or diluent,respectively.

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to a molecular chain of two or more amino acids linked throughpeptide bonds. The terms do not refer to a specific length of theproduct. Thus, “peptides,” and “oligopeptides,” are included within thedefinition of polypeptide. The terms include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. The terms also includemolecules in which one or more amino acid analogs or non-canonical orunnatural amino acids are included as can be synthesized, or expressedrecombinantly using known protein engineering techniques. In addition,proteins can be derivatized as described herein by well-known organicchemistry techniques.

The term “primary T-cell assay” as used herein refers to an in vitroassay to measure interferon-gamma (“IFN-gamma”) expression. A variety ofsuch primary T-cell assays are known in the art such as that describedin Example 6. In a preferred embodiment, the assay used is anti-CD3coimmobilization assay. In this assay, primary T cells are stimulated byanti-CD3 immobilized with or without additional recombinant proteins.Culture supernatants are harvested at timepoints, usually 24-72 hours.In another embodiment, the assay used is a mixed lymphocyte reaction(MLR). In this assay, primary T cells are simulated with allogenic APC.Culture supernatants are harvested at timepoints, usually 24-72 hours.Human IFN-gamma levels are measured in culture supernatants by standardELISA techniques. Commercial kits are available from vendors and theassay is performed according to manufacturer's recommendation.

The term “purified” as applied to nucleic acids or immunomodulatoryproteins of the invention generally denotes a nucleic acid orpolypeptide that is substantially free from other components asdetermined by analytical techniques well known in the art (e.g., apurified polypeptide or polynucleotide forms a discrete band in anelectrophoretic gel, chromatographic eluate, and/or a media subjected todensity gradient centrifugation). For example, a nucleic acid orpolypeptide that gives rise to essentially one band in anelectrophoretic gel is “purified.” A purified nucleic acid orimmunomodulatory protein of the invention is at least about 50% pure,usually at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure(e.g., percent by weight or on a molar basis).

The term “recombinant” indicates that the material (e.g., a nucleic acidor a polypeptide) has been artificially (i.e., non-naturally) altered byhuman intervention. The alteration can be performed on the materialwithin, or removed from, its natural environment or state. For example,a “recombinant nucleic acid” is one that is made by recombining nucleicacids, e.g., during cloning, affinity modification, DNA shuffling orother well-known molecular biological procedures. A “recombinant DNAmolecule,” is comprised of segments of DNA joined together by means ofsuch molecular biological techniques. The term “recombinant protein” or“recombinant polypeptide” as used herein refers to a protein molecule(e.g., an immunomodulatory protein) which is expressed using arecombinant DNA molecule. A “recombinant host cell” is a cell thatcontains and/or expresses a recombinant nucleic acid. Transcriptionalcontrol signals in eukaryotes comprise “promoter” and “enhancer”elements. Promoters and enhancers consist of short arrays of DNAsequences that interact specifically with cellular proteins involved intranscription. Promoter and enhancer elements have been isolated from avariety of eukaryotic sources including genes in yeast, insect andmammalian cells and viruses (analogous control elements, i.e.,promoters, are also found in prokaryotes). The selection of a particularpromoter and enhancer depends on what cell type is to be used to expressthe protein of interest. The terms “in operable combination,” “inoperable order” and “operably linked” as used herein refer to thelinkage of nucleic acid sequences in such a manner or orientation that anucleic acid molecule capable of directing the transcription of a givengene and/or the synthesis of a desired protein molecule is produced. Theterm also refers to the linkage of amino acid sequences in such a mannerso that a functional protein is produced and/or transported.

The term “recombinant expression vector” as used herein refers to a DNAmolecule containing a desired coding sequence (e.g., an immunomodulatorynucleic acid) and appropriate nucleic acid sequences necessary for theexpression of the operably linked coding sequence in a particular hostcell. Nucleic acid sequences necessary for expression in prokaryotesinclude a promoter, optionally an operator sequence, a ribosome bindingsite and possibly other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals. Asecretory signal peptide sequence can also, optionally, be encoded bythe recombinant expression vector, operably linked to the codingsequence for the inventive recombinant fusion protein, so that theexpressed fusion protein can be secreted by the recombinant host cell,for more facile isolation of the fusion protein from the cell, ifdesired.

The term “sequence identity” as used herein refers to the sequenceidentity between genes or proteins at the nucleotide or amino acidlevel, respectively. “Sequence identity” is a measure of identitybetween proteins at the amino acid level and a measure of identitybetween nucleic acids at nucleotide level. The protein sequence identitymay be determined by comparing the amino acid sequence in a givenposition in each sequence when the sequences are aligned. Similarly, thenucleic acid sequence identity may be determined by comparing thenucleotide sequence in a given position in each sequence when thesequences are aligned. Methods for the alignment of sequences forcomparison are well known in the art, such methods include GAP, BESTFIT,BLAST, FASTA and TFASTA. The BLAST algorithm calculates percent sequenceidentity and performs a statistical analysis of the similarity betweenthe two sequences. The software for performing BLAST analysis ispublicly available through the National Center for BiotechnologyInformation (NCBI) website.

The term “soluble” as used herein in reference to proteins, means thatthe protein is not a membrane protein. In general, a soluble proteincontains only the extracellular domain of an IgSF family memberreceptor, or a portion thereof containing an IgSF domain or domains orspecific-binding fragments thereof.

The term “species” as used herein in the context of a nucleic acidsequence or a polypeptide sequence refers to an identical collection ofsuch sequences. Slightly truncated sequences that differ (or encode adifference) from the full length species at the amino-terminus orcarboxy-terminus by no more than 1, 2, or 3 amino acid residues areconsidered to be of a single species. Such microheterogeneities are acommon feature of manufactured proteins.

The term “specifically binds” as used herein means the ability of aprotein, under specific binding conditions, to bind to a target proteinsuch that its affinity or avidity is at least 10 times as great, butoptionally 50, 100, 250 or 500 times as great, or even at least 1000times as great as the average affinity or avidity of the same protein toa collection of random peptides or polypeptides of sufficientstatistical size. A specifically binding protein need not bindexclusively to a single target molecule (e.g., its cognate bindingpartner) but may specifically bind to a non-target molecule due tosimilarity in structural conformation between the target and non-target(e.g., paralogs or orthologs). Those of skill will recognize thatspecific binding to a molecule having the same function in a differentspecies of animal (i.e., ortholog) or to a non-target molecule having asubstantially similar epitope as the target molecule (e.g., paralog) ispossible and does not detract from the specificity of binding which isdetermined relative to a statistically valid collection of uniquenon-targets (e.g., random polypeptides). Thus, an affinity modifiedpolypeptide of the invention may specifically bind to more than onedistinct species of target molecule due to cross-reactivity. Generally,such off-target specific binding is mitigated by reducing affinity oravidity for undesired targets. Solid-phase ELISA immunoassays or Biacoremeasurements can be used to determine specific binding between twoproteins. Generally, interactions between two binding proteins havedissociation constants (Kd) less than 1×10⁻⁵ M, and often as low as1×10⁻¹² M. In certain aspects of the present disclosure, interactionsbetween two binding proteins have dissociation constants of 1×10⁻⁶ M,1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M or 1×10⁻¹¹ M.

The term “specific binding fragment” or “fragment” as used herein inreference to a mature (i.e., absent the signal peptide) wild-type IgSFdomain, means a polypeptide that is shorter than the full-length matureIgSF domain and that specifically binds in vitro and/or in vivo to themature wild-type IgSF domain's native cognate binding partner. In someembodiments, the specific binding fragment is at at least 20%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% thesequence length of the full-length mature wild-type sequence. Thespecific binding fragment can be altered in sequence to form an affinitymodified IgSF domain of the invention. In some embodiments, the specificbinding fragment modulates immunological activity of a lymphocyte.

The terms “suppressed” or “decreased” as used herein means to decreaseby a statistically significant amount. In some embodiments suppressioncan be a decrease of at least 10%, and up to 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%.

The term “targeting moiety” as used herein refers to a composition thatis covalently or non-covalently attached to, or physically encapsulates,a polypeptide comprising a wild-type and/or affinity modified IgSFdomain of the present invention. The targeting moiety has specificbinding affinity for a desired cognate binding partner such as a cellsurface receptor or a tumor antigen such as tumor specific antigen (TSA)or a tumor associated antigen (TAA). Typically, the desired cognatebinding partner is localized on a specific tissue or cell-type.Targeting moieties include: antibodies, antigen binding fragment (Fab),variable fragment (Fv) containing V_(H) and V_(L), the single chainvariable fragment (scFv) containing V_(H) and V_(L) linked together inone chain, as well as other antibody V region fragments, such as Fab′,F(ab)₂, F(ab′)₂, dsFv diabody, nanobodies, soluble receptors, receptorligands, affinity matured receptors or ligands, as well as smallmolecule (<500 dalton) compositions (e.g., specific binding receptorcompositions). Targeting moieties can also be attached covalently ornon-covalently to the lipid membrane of liposomes that encapsulate animmunomodulatory polypeptide of the present invention.

The terms “treating,” “treatment,” or “therapy” of a disease or disorderas used herein mean slowing, stopping or reversing the disease ordisorders progression, as evidenced by decreasing, cessation orelimination of either clinical or diagnostic symptoms, by administrationof an immunomodulatory protein of the present invention either alone orin combination with another compound as described herein. “Treating,”“treatment,” or “therapy” also means a decrease in the severity ofsymptoms in an acute or chronic disease or disorder or a decrease in therelapse rate as for example in the case of a relapsing or remittingautoimmune disease course or a decrease in inflammation in the case ofan inflammatory aspect of an autoimmune disease. As used herein in thecontext of cancer, the terms “treatment” or, “inhibit,” “inhibiting” or“inhibition” of cancer refers to at least one of: a statisticallysignificant decrease in the rate of tumor growth, a cessation of tumorgrowth, or a reduction in the size, mass, metabolic activity, or volumeof the tumor, as measured by standard criteria such as, but not limitedto, the Response Evaluation Criteria for Solid Tumors (RECIST), or astatistically significant increase in progression free survival (PFS) oroverall survival (OS). “Preventing,” “prophylaxis,” or “prevention” of adisease or disorder as used in the context of this invention refers tothe administration of an immunomodulatory protein of the presentinvention, either alone or in combination with another compound, toprevent the occurrence or onset of a disease or disorder or some or allof the symptoms of a disease or disorder or to lessen the likelihood ofthe onset of a disease or disorder.

The term “tumor specific antigen” or “TSA” as used herein refers to anantigen that is present primarily on tumor cells of a mammalian subjectbut generally not found on normal cells of the mammalian subject. Atumor specific antigen need not be exclusive to tumor cells but thepercentage of cells of a particular mammal that have the tumor specificantigen is sufficiently high or the levels of the tumor specific antigenon the surface of the tumor are sufficiently high such that it can betargeted by anti-tumor therapeutics, such as immunomodulatorypolypeptides of the invention, and provide prevention or treatment ofthe mammal from the effects of the tumor. In some embodiments, in arandom statistical sample of cells from a mammal with a tumor, at least50% of the cells displaying a TSA are cancerous. In other embodiments,at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells displaying aTSA are cancerous.

As used herein, “screening” refers to identification or selection of amolecule or portion thereof from a collection or library of moleculesand/or portions thereof, based on determination of the activity orproperty of a molecule or portion thereof. Screening can be performed inany of a variety of ways, including, for example, by assays assessingdirect binding (e.g. binding affinity) of the molecule to a targetprotein or by functional assays assessing modulation of an activity of atarget protein.

The term “wild-type” or “natural” or “native” or “parental” as usedherein is used in connection with biological materials such as nucleicacid molecules, proteins, IgSF members, host cells, and the like, refersto those which are found in nature and not modified by humanintervention. A wild-type IgSF domain is a type of non-affinity modifiedIgSF domain. In some embodiments of immunomodulatory proteins of theinvention, a non-affinity modified IgSF is a wild-type IgSF domain.

II. Affinity-Modified Immunomodulatory Proteins

The present invention provides immunomodulatory proteins that havetherapeutic utility by modulating immunological activity in a mammalwith a disease or disorder in which modulation of the immune systemresponse is beneficial.

The IgSF family members included within the scope of theimmunomodulatory proteins of the present invention excludes antibodies(i.e., immunoglobulins) such as those that are mammalian or may be ofmammalian origin. Thus, the present invention relates tonon-immunoglobulin (i.e., non-antibody) IgSF domains. Wild-typemammalian IgSF family members that are not immunoglobulins (i.e.antibodies) are known in the art as are their nucleic and amino acidsequences. All non-immunoglobulin mammalian IgSF family members areincluded within the scope of the invention.

In some embodiments, the non-immunoglobulin IgSF family members, and thecorresponding IgSF domains present therein, are of mouse, rat,cynomolgus monkey, or human origin. In some embodiments, the IgSF familymembers are members from at least or exactly one, two, three, four,five, or more IgSF subfamilies such as: Signal-Regulatory Protein (SIRP)Family, Triggering Receptor Expressed On Myeloid Cells Like (TREML)Family, Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM)Family, Sialic Acid Binding Ig-Like Lectin (SIGLEC) Family, ButyrophilinFamily, B7 family, CD28 family, V-set and Immunoglobulin DomainContaining (VSIG) family, V-set transmembrane Domain (VSTM) family,Major Histocompatibility Complex (MHC) family, Signaling lymphocyticactivation molecule (SLAM) family, Leukocyte immunoglobulin-likereceptor (LIR), Nectin (Nec) family, Nectin-like (NECL) family,Poliovirus receptor related (PVR) family, Natural cytotoxicitytriggering receptor (NCR) family, or Killer-cell immunoglobulin-likereceptors (KIR) family.

In some embodiments, non-immunoglobulin IgSF family members, and thecorresponding IgSF domains present therein, of an immunomodulatoryprotein of the invention, are affinity-modified compared to a mammalianIgSF member. In some embodiments, the mammalian IgSF member is one ofthe IgSF members or comprises an IgSF domain from one of the IgSFmembers as indicated in Table 1 including any mammalian orthologsthereof. Orthologs are genes in different species that evolved from acommon ancestral gene by speciation. Normally, orthologs retain the samefunction in the course of evolution.

The first column of Table 1 provides the name and, optionally, the nameof some possible synonyms for that particular IgSF member. The secondcolumn provides the protein identifier of the UniProtKB database, apublicly available database accessible via the internet at uniprot.org.The Universal Protein Resource (UniProt) is a comprehensive resource forprotein sequence and annotation data. The UniProt databases include theUniProt Knowledgebase (UniProtKB). UniProt is a collaboration betweenthe European Bioinformatics Institute (EMBL-EBI), the SIB SwissInstitute of Bioinformatics and the Protein Information Resource (PIR)and supported mainly by a grant from the U.S. National Institutes ofHealth (NIH). The third column provides the region where the indicatedIgSF domain is located. The region is specified as a range where thedomain is inclusive of the residues defining the range. Column 3 alsoindicates the IgSF domain class for the specified IgSF region. Column 4provides the region where the indicated additional domains are located(signal peptide, S; extracellular domain, E; transmembrane domain, T;cytoplasmic domain, C). Column 5 indicates for some of the listed IgSFmembers, some of its cognate cell surface binding partners.

Typically, the affinity modified IgSF domain of the provided embodimentsis a human or murine affinity modified IgSF domain.

TABLE 1 IgSF members according to the present disclosure. IgSF MemberAmino Acid Cognate Cell Sequence (SEQ ID NO) IgSF UniProtKB SurfacePrecursor Member Protein IgSF Region & Other Binding (mature (Synonyms)Identifier Domain Class Domains Partners residues) ECD CD80 NP_005182.135-138 or S: 1-34, CD28, CTLA4, SEQ ID NO: 1 SEQ ID NO: 28 (B7-1) P3368137-138 IgV, E: 35-242, PD-L1 (35-288) 145-230 or T: 243-263, 154-232 IgCC: 264-288 CD86 P42081.2 33-131 IgV, S: 1-23, CD28, CTLA4 SEQ ID NO: 2SEQ ID NO: 29 (B7-2) 150-225 IgC2 E: 24-247, (24-329) T: 248-268, C:269-329 CD274 Q9NZQ7.1 24-130 IgV, S: 1-18, PD-1, B7-1 SEQ ID NO: 3 SEQID NO: 30 (PD-L1, 133-225 IgC2 E: 19-238, (19-290) B7-H1) T: 239-259, C:260-290 PDCD1LG2 Q9BQ51.2 21-118 IgV, S: 1-19, PD-1, RGMb SEQ ID NO: 4SEQ ID NO: 31 (PD-L2, 122-203 IgC2 E: 20-220, (20-273) CD273) T:221-241, C: 242-273 ICOSLG O75144.2 19-129 IgV, S: 1-18, ICOS, CD28, SEQID NO: 5 SEQ ID NO: 32 (B7RP1, 141-227 IgC2 E: 19-256, CTLA4 (19-302)CD275, T: 257-277, ICOSL, C: 278-302 B7-H2) CD276 Q5ZPR3.1 29-139 IgV,S: 1-28, SEQ ID NO: 6 SEQ ID NO: 33 (B7-H3) 145-238 IgC2, E: 29-466,(29-534) 243-357 IgV, T: 467-487, 367-453 IgC C: 488-534 VTCN1 Q7Z7D3.135-146 IgV, S: 1-24, SEQ ID NO: 7 SEQ ID NO: 34 (B7-H4) 153-241 IgV E:25-259, (25-282) T: 260-280, C: 281-282 CD28 P10747.1 28-137 IgV S:1-18, B7-1, B7-2, SEQ ID NO: 8 SEQ ID NO: 35 E: 19-152, B7RP1 (19-220)T: 153-179, C: 180-220 CTLA4 P16410.3 39-140 IgV S: 1-35, B7-1, B7-2,SEQ ID NO: 9 SEQ ID NO: 36 E: 36-161, B7RP1 (36-223) T: 162-182, C:183-223 PDCD1 Q15116.3 35-145 IgV S: 1-20, PD-L1, PD-L2 SEQ ID NO: 10SEQ ID NO: 37 (PD-1) E: 21-170, (21-288) T: 171-191, C: 192-288 ICOSQ9Y6W8.1 30-132 IgV S: 1-20, B7RP1 SEQ ID NO: 11 SEQ ID NO: 38 E:21-140, (21-199) T: 141-161, C: 162-199 BTLA Q7Z6A9.3 31-132 IgV S:1-30, HVEM SEQ ID NO: 12 SEQ ID NO: 39 (CD272) E: 31-157, (31-289) T:158-178, C: 179-289 CD4 P01730.1 26-125 IgV, S: 1-25, MHC class II SEQID NO: 13 SEQ ID NO: 40 126-203 IgC2, E: 26-396, (26-458) 204-317 IgC2,T: 397-418, 317-389 IgC2 C: 419-458 CD8A P01732.1 22-135 IgV S: 1-21,MHC class I SEQ ID NO: 14 SEQ ID NO: 41 (CD8-alpha) E: 22-182, (22-235)T: 183-203, C: 204-235 CD8B P10966.1 22-132 IgV S: 1-21, MHC class I SEQID NO: 15 SEQ ID NO: 42 (CD8-beta) E: 22-170, (22-210) T: 171-191, C:192-210 LAG3 P18627.5 37-167 IgV, S: 1-28, MHC class II SEQ ID NO: 16SEQ ID NO: 43 168-252 IgC2, E: 29-450, (29-525) 265-343 IgC2, T:451-471, 349-419 IgC2 C: 472-525 HAVCR2 Q8TDQ0.3 22-124 IgV S: 1-21,CEACAM-1, SEQ ID NO: 17 SEQ ID NO: 44 (TIM-3) E: 22-202,phosphatidylserine, (22-301) T: 203-223, Galectin-9, C: 224-301 HMGB1CEACAM1 P13688.2 35-142 IgV, S: 1-34, TIM-3 SEQ ID NO: 18 SEQ ID NO: 45145-232 IgC2, E: 35-428, (35-526) 237-317 IgC2, T: 429-452, 323-413 IgCC: 453-526 TIGIT Q495A1.1 22-124 IgV S: 1-21, CD155, CD112 SEQ ID NO: 19SEQ ID NO: 46 E: 22-141, (22-244) T: 142-162, C: 163-244 PVR P15151.224-139 IgV, S: 1-20, TIGIT, CD226, SEQ ID NO: 20 SEQ ID NO: 47 (CD155)145-237 IgC2, E: 21-343, CD96, poliovirus (21-417) 244-328 IgC2 T:344-367, C: 368-417 PVRL2 Q92692.1 32-156 IgV, S: 1-31, TIGIT, CD226,SEQ ID NO: 21 SEQ ID NO: 48 (CD112) 162-256 IgC2, E: 32-360, CD112R(32-538) 261-345 IgC2 T: 361-381, C: 382-538 CD226 Q15762.2 19-126 IgC2,S: 1-18, CD155, CD112 SEQ ID NO: 22 SEQ ID NO: 49 135-239 IgC2 E:19-254, (19-336) T: 255-275, C: 276-336 CD2 P06729.2 25-128 IgV, S:1-24, CD58 SEQ ID NO: 23 SEQ ID NO: 50 129-209 IgC2 E: 25-209, (25-351)T: 210-235, C: 236-351 CD160 O95971.1 27-122 IgV N/A HVEM, MHC SEQ IDNO: 24 SEQ ID NO: 51 family of proteins (27-159) CD200 P41217.4 31-141IgV, S: 1-30, CD200R SEQ ID NO: 25 SEQ ID NO: 52 142-232 IgC2 E: 31-232,(31-278) T: 233-259, C: 260-278 CD200R1 Q8TD46.2 53-139 IgV, S: 1-28,CD200 SEQ ID NO: 26 SEQ ID NO: 53 (CD200R) 140-228 IgC2 E: 29-243,(29-325) T: 244-264, C: 265-325 NCR3 O14931.1 19-126 IgC-like S: 1-18,B7-H6 SEQ ID NO: 27 SEQ ID NO: 54 (NKp30) E: 19-135, (19-201) T:136-156, C: 157-201

In some embodiments, the immunomodulatory proteins of the presentinvention comprise at least one affinity modified mammalian IgSF domain.The affinity modified IgSF domain can be affinity modified tospecifically bind to a single or multiple (2, 3, 4, or more) cognatebinding partners (also called a “counter structure ligand”). An IgSFdomain can be affinity modified to independently increase or decreasespecific binding affinity or avidity to each of the multiple cognatebinding partners to which it binds. By this mechanism, specific bindingto each of multiple cognate binding partners is independently tuned to aparticular affinity or avidity.

In some embodiments, the cognate binding partner of an IgSF domain is atleast one, and sometimes at least two or three of the cognate bindingpartners of the wild-type IgSF domain, such as those listed in Table 1.The sequence of the IgSF domain, such as a mammalian IgSF domain, isaffinity modified by altering its sequence with at least onesubstitution, addition, or deletion. Alteration of the sequence canoccur at the binding site for the cognate binding partner or at anallosteric site. In some embodiments, a nucleic acid encoding a IgSFdomain, such as a mammalian IgSF domain, is affinity modified bysubstitution, addition, deletion, or combinations thereof, of specificand pre-determined nucleotide sites to yield a nucleic acid of theinvention. In some contrasting embodiments, a nucleic acid encoding anIgSF domain, such as a mammalian IgSF domain, is affinity modified bysubstitution, addition, deletion, or combinations thereof, at randomsites within the nucleic acid. In some embodiments, a combination of thetwo approaches (pre-determined and random) is utilized. In someembodiments, design of the affinity modified IgSF domains of the presentinvention is performed in silico.

In some embodiments, the affinity modified IgSF domain contains one ormore amino acid substitutions (alternatively, “mutations” or“replacements”) relative to a wild-type or unmodified polypeptide or aportion thereof containing an immunoglobulin superfamily (IgSF) domain,such as an IgV domain or an IgC domain or specific binding fragment ofthe IgV domain or the IgC domain. In some embodiments, theimmunomodulatory protein comprises an affinity modified IgSF domain thatcontains an IgV domain or an IgC domain or specific binding fragmentsthereof in which the at least one of the amino acid substitutions is inthe IgV domain or IgC domain or a specific binding fragment thereof. Insome embodiments, by virtue of the altered binding activity or affinity,the IgV domain or IgC domain is an affinity-modified IgSF domain.

In some embodiments, the IgSF domain, such as a mammalian IgSF domain,is affinity modified in sequence with at least one but no more than atotal of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof. In some embodiments, theIgSF domain, such as a mammalian IgSF domain, is affinity modified insequence with at least one but no more than 10, 9, 8, 7, 6, 5, 4, 3, or2 amino acid substitutions. In some embodiments, the substitutions areconservative substitutions. In some embodiments, the substitutions arenon-conservative. In some embodiments, the substitutions are acombination of conservative and non-conservative substitutions. In someembodiments, the modification in sequence is made at the binding site ofthe IgSF domain for its cognate binding partner.

In some embodiments, the wild-type or unmodified IgSF domain is amammalian IgSF domain. In some embodiments, the wild-type or unmodifiedIgSF domain can be an IgSF domain that includes, but is not limited to,human, mouse, cynomolgus monkey, or rat. In some embodiments, thewild-type or unmodified IgSF domain is human.

In some embodiments, the wild-type or unmodified IgSF domain is an IgSFdomain or specific binding fragment thereof contained in the sequence ofamino acids set forth in any of SEQ ID NO:1-27 or a mature form thereoflacking the signal sequence, a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO:1-27 or a mature formthereof, or is a portion thereof containing an IgV domain or IgC domainor specific binding fragments thereof.

In some embodiments, the wild-type or unmodified IgSF domain is orcomprises an extracellular domain of an IgSF family member or a portionthereof containing an IgSF domain (e.g. IgV domain or IgC domain). Insome embodiments, the unmodified or wild-type IgSF domain comprises theamino acid sequence set forth in any of SEQ ID NOS:28-54, or an orthologthereof. For example, the unmodified or wild-type IgSF domain cancomprise (i) the sequence of amino acids set forth in any of SEQ IDNOS:28-54, (ii) a sequence of amino acids that has at least about 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity to any of SEQ ID NOS: 28-54, or (iii) is a specificbinding fragment of the sequence of amino acids set forth in any of SEQID NOS: 28-54 or a specific binding fragment of a sequence of aminoacids that has at least at least about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any ofSEQ ID NOS: 28-54 comprising an IgV domain or an IgC domain.

In some embodiments, the extracellular domain of an unmodified orwild-type IgSF domain can comprise more than one IgSF domain, forexample, an IgV domain and an IgC domain. However, the affinity modifiedIgSF domain need not comprise both the IgV domain and the IgC domain. Insome embodiments, the affinity modified IgSF domain comprises orconsists essentially of the IgV domain or a specific binding fragmentthereof. In some embodiments, the affinity modified IgSF domaincomprises or consists essentially of the IgC domain or a specificbinding fragment thereof. In some embodiments, the affinity modifiedIgSF domain comprises the IgV domain or a specific binding fragmentthereof, and the IgC domain or a specific binding fragment thereof.

In some embodiments, the one or more amino acid substitutions of theaffinity modified IgSF domain can be located in any one or more of theIgSF polypeptide domains. For example, in some embodiments, one or moreamino acid substitutions are located in the extracellular domain of theIgSF polypeptide. In some embodiments, one or more amino acidsubstitutions are located in the IgV domain or specific binding fragmentof the IgV domain. In some embodiments, one or more amino acidsubstitutions are located in the IgC domain or specific binding fragmentof the IgC domain.

In some embodiments, at least one IgSF domain, such as a mammalian IgSFdomain, of an immunomodulatory protein provided herein is independentlyaffinity modified in sequence to have at least 99%, 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86% 85%, or 80% sequenceidentity with a wild-type or unmodified IgSF domain or specific bindingfragment thereof contained in a wild-type or unmodified IgSF protein,such as but not limited to, those disclosed in Table 1 as SEQ ID NOS:1-27.

In some embodiments, the IgSF domain of an immunomodulatory proteinprovided herein is a specific binding fragment of a wild-type orunmodified IgSF domain contained in a wild-type or unmodified IgSFprotein, such as but not limited to, those disclosed in Table 1 in SEQID NOS: 1-27. In some embodiments, the specific binding fragment canhave an amino acid length of at least 50 amino acids, such as at least60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, thespecific binding fragment of the IgV domain contains an amino acidsequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% of the length of the wild-type orunmodified IgV domain. In some embodiments, the specific bindingfragment of the IgC domain comprises an amino acid sequence that is atleast about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% of the length of the wild-type or unmodified IgC domain.In some embodiments, the specific binding fragment modulatesimmunological activity. In more specific embodiments, the specificbinding fragment of an IgSF domain increases immunological activity. Inalternative embodiments, the specific binding fragment decreasesimmunological activity.

To determine the percent identity of two nucleic acid sequences or oftwo amino acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps may be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength. One may manually align the sequences and count the number ofidentical nucleic acids or amino acids. Alternatively, alignment of twosequences for the determination of percent identity may be accomplishedusing a mathematical algorithm. Such an algorithm is incorporated intothe NBLAST and XBLAST programs. BLAST nucleotide searches may beperformed with the NBLAST program, score=100, wordlength=12, to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches may be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecule of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST may be utilized.Alternatively, PSI-Blast may be used to perform an iterated search whichdetects distant relationships between molecules. When utilising theNBLAST, XBLAST, and Gapped BLAST programs, the default parameters of therespective programs may be used such as those available on the NCBIwebsite. Alternatively, sequence identity may be calculated after thesequences have been aligned e.g. by the BLAST program in the NCBIdatabase. Generally, the default settings with respect to e.g. “scoringmatrix” and “gap penalty” may be used for alignment. In the context ofthe present invention, the BLASTN and PSI BLAST NCBI default settingsmay be employed.

In some embodiments, the immunomodulatory protein contains at least oneaffinity modified IgSF domain. In some embodiments, the immunomodulatoryprotein further contains at least one affinity modified domain andfurther contains at least one non-affinity modified IgSF domain (e.g.unmodified or wildtype IgSF domain). In some embodiments, theimmunomodulatory protein contains at least two affinity modifieddomains. In some embodiments, the immunomodulatory protein can contain aplurality of non-affinity modified IgSF domains and/or affinity modifiedIgSF domains such as 1, 2, 3, 4, 5, or 6 non-affinity modified IgSFand/or affinity modified IgSF domains.

In some embodiments, at least one non-affinity modified IgSF domainand/or one affinity modified IgSF domain present in an immunomodulatoryprotein provided herein specifically binds to at least one cell surfacemolecular species expressed on mammalian cells forming the immunologicalsynapse (IS). Of course, in some embodiments, an immunomodulatoryprotein provided herein comprises a plurality of non-affinity modifiedIgSF domains and/or affinity modified IgSF domains such as 1, 2, 3, 4,5, or 6 non-affinity modified IgSF and/or affinity modified IgSFdomains. One or more of these non-affinity modified IgSF domains and/oraffinity modified IgSF domains can independently specifically bind toeither one or both of the mammalian cells forming the IS.

Often, the cell surface molecular species to which the affinity modifiedIgSF domain specifically binds will be the cognate binding partner ofthe wild type IgSF family member or wild type IgSF domain that has beenaffinity modified. In some embodiments, the cell surface molecularspecies is a mammalian IgSF member. In some embodiments, the cellsurface molecular species is a human IgSF member. In some embodiments,the cell surface molecular species will be the cell surface cognatebinding partners as indicated in Table 1. In some embodiments, the cellsurface molecular species will be a viral protein, such as a poliovirusprotein, on the cell surface of a mammalian cell such as a human cell.

In some embodiments, at least one non-affinity modified and/or affinitymodified IgSF domain of an immunomodulatory protein provided hereinbinds to at least two or three cell surface molecular species present onmammalian cells forming the IS. The cell surface molecular species towhich the non-affinity modified IgSF domains and/or the affinitymodified IgSF domains of the invention specifically bind to canexclusively be on one or the other of the two mammalian cells (i.e. incis configuration) forming the IS or, alternatively, the cell surfacemolecular species can be present on both.

In some embodiments, the affinity modified IgSF domain specificallybinds to at least two cell surface molecular species wherein one of themolecular species is present on one of the two mammalian cells formingthe IS and the other molecular species is present on the second of thetwo mammalian cells forming the IS. In such embodiments, the cellsurface molecular species is not necessarily present solely on one orthe other of the two mammalian cells forming the IS (i.e., in a transconfiguration) although in some embodiments it is. Thus, embodimentsprovided herein include those wherein each cell surface molecularspecies is exclusively on one or the other of the mammalian cellsforming the IS (cis configuration) as well as those where the cellsurface molecular species to which each affinity modified IgSF binds ispresent on both of the mammalian cells forming the IS (i.e., cis andtrans configuration).

Those of skill will recognize that antigen presenting cells (APCs) andtumor cells form an immunological synapse with lymphocytes. Thus, insome embodiments at least one non-affinity modified IgSF domain and/orat least one affinity modified IgSF domain of the immunomodulatoryprotein specifically binds to only cell surface molecular speciespresent on a cancer cell, wherein the cancer cell in conjunction with alymphocyte forms the IS. In other embodiments, at least one non-affinitymodified IgSF domain and/or at least one affinity modified IgSF domainof the immunomodulatory protein specifically binds to only cell surfacemolecular species present on a lymphocyte, wherein the lymphocyte inconjunction with an APC or tumor cell forms the IS. In some embodiments,the non-affinity modified IgSF domain and/or affinity modified IgSFdomain bind to cell surface molecular species present on both the targetcell (or APC) and the lymphocyte forming the IS.

Embodiments of the invention include those in which an immunomodulatoryprotein provided herein comprises at least one affinity modified IgSFdomain with an amino acid sequence that differs from a wild-type orunmodified IgSF domain (e.g. a mammalian IgSF domain) such that itsbinding affinity (or avidity if in a multimeric or other relevantstructure), under specific binding conditions, to at least one of itscognate binding partners is either increased or decreased relative tothe unaltered wild-type or unmodified IgSF domain control. In someembodiments, an affinity modified IgSF domain has a binding affinity fora cognate binding partner that differs from that of a wild-type orunmodified IgSF control sequence as determined by, for example,solid-phase ELISA immunoassays, flow cytometry or Biacore assays. Insome embodiments, the IgSF domain has an increased binding affinity forone or more cognate binding partners. In some embodiments, the affinitymodified IgSF domain has a decreased binding affinity for one or morecognate binding partners, relative to a wild-type or unmodified IgSFdomain. In some embodiments, the cognate binding partner can be amammalian protein, such as a human protein or a murine protein.

Binding affinities for each of the cognate binding partners areindependent; that is, in some embodiments, an affinity modified IgSFdomain has an increased binding affinity for one, two or three differentcognate binding parters, and a decreased binding affinity for one, twoor three of different cognate binding partners, relative to a wild-typeor unmodified ICOSL polypeptide.

In some embodiments of an immunomodulatory protein provided herein, thebinding affinity or avidity of the affinity modified IgSF domain isincreased at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%,500%, 1000%, 5000%, or 10,000% relative to the wild type or unmodifiedcontrol IgSF domain. In some embodiments, the increase in bindingaffinity relative to the wild-type or unmodified IgSF domain is morethan 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold.

In some embodiments, the binding affinity or avidity is decreased atleast 10%, and up to 20%, 30%, 40%, 50%, 60%, 70%, 80% or up to 90%relative to the wild type or unmodified control IgSF domain. In someembodiments, the decrease in binding affinity relative to the wild-typeor unmodified IgSF domain is more than 1.2-fold, 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold 40-fold or 50-fold.

In some embodiments, the specific binding affinity of an affinitymodified IgSF domain to a cognate binding partner can be at least 1×10⁻⁵M, 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M or 1×10⁻¹¹M, or1×10⁻¹² M.

In some embodiments, an immunomodulatory protein provided comprises atleast two IgSF domains in which at least one of the IgSF domain isaffinity modified while in some embodiments both are affinity modified,and wherein at least one of the affinity modified IgSF domains hasincreased affinity (or avidity) to its cognate binding partner and atleast one affinity modified IgSF domain has a decreased affinity (oravidity) to its cognate binding partner.

In some embodiments, an IgSF domain that otherwise binds to multiplecell surface molecular species is affinity modified such that itsubstantially no longer specifically binds to one of its cognate cellsurface molecular species. Thus, in these embodiments the specificbinding to one of its cognate cell surface molecular species is reducedto specific binding of no more than 10% of the wild type level and oftenno more than 7%, 5%, 3%, 1%, or no detectable or statisticallysignificant specific binding.

In these embodiments, a specific binding site on a mammalian IgSF domainis inactivated or substantially inactivated with respect to at least oneof the cell surface molecular species. Thus, for example, if a wild typeIgSF domain specifically binds to exactly two cell surface molecularspecies then in some embodiments it is affinity modified to specificallybind to exactly one cell surface molecular species (wherebydetermination of the number of affinity modified IgSF domains disregardsany substantially immunologically inactive fractional sequence thereof).And, if a wild type IgSF domain specifically binds to exactly three cellsurface molecular species then in some embodiments it is affinitymodified to specifically bind to exactly two cell surface molecularspecies. The IgSF domain that is affinity modified to substantially nolonger specifically bind to one of its cognate cell surface molecularspecies can be an IgSF domain that otherwise specifically bindscompetitively or non-competitively to its cell surface molecularspecies. Those of skill will appreciate that a wild type IgSF domainthat competitively binds to two cognate binding partners can nonethelessbe inactivated with respect to exactly one of them if, for example,their binding sites are not not precisely coextensive but merely overlapsuch that specific binding of one inhibits binding of the other cognatebinding partner and yet both competitive binding sites are distinct.

The non-affinity modified IgSF domains and/or affinity modified IgSFdomains of the immunomodulatory proteins provided can in someembodiments specifically bind competitively to its cognate cell surfacemolecular species. In other embodiments the non-affinity modified IgSFdomains and/or affinity modified IgSF domains of an immunomodulatoryprotein provided herein specifically bind non-competitively to itscognate cell surface molecular species. Any number of the non-affinitymodified IgSF domains and/or affinity modified IgSF domains present inan immunomodulatory protein provided herein can specifically bindcompetitively or non-competitively.

In some embodiments, the immunomodulatory protein provided hereincomprises at least two non-affinity modified IgSF domains, or at leastone non-affinity modified IgSF domain and at least one affinity modifiedIgSF domain, or at least two affinity modified IgSF domains wherein oneIgSF domain specifically binds competitively and a second IgSF domainbinds non-competitively to its cognate cell surface molecular species.More generally, an immunomodulatory protein provided herein can comprise1, 2, 3, 4, 5, or 6 competitive or 1, 2, 3, 4, 5, or 6 non-competitivebinding non-affinity modified IgSF and/or affinity modified IgSF domainsor any combination thereof. Thus, an immunomodulatory protein providedherein can have the number of non-competitive and competitive bindingIgSF domains, respectively, of: 0 and 1, 0 and 2, 0 and 3, 0 and 4, 1and 0, 1 and 1, 1 and 2, 1 and 3, 2 and 0, 2 and 1, 2 and 2, 2 and 3, 3and 0, 3 and 1, 3 and 2, 3 and 3, 4 and 0, 4 and 1, and, 4 and 2.

A plurality of non-affinity modified and/or affinity modified IgSFdomains immunomodulatory protein provided herein need not be covalentlylinked directly to one another. In some embodiments, an intervening spanof one or more amino acid residues indirectly covalently bonds thenon-affinity modified and/or affinity modified IgSF domains to eachother. The linkage can be via the N-terminal to C-terminal residues.

In some embodiments, the linkage can be made via side chains of aminoacid residues that are not located at the N-terminus or C-terminus ofthe non-affinity modified or affinity modified IgSF domain. Thus,linkages can be made via terminal or internal amino acid residues orcombinations thereof.

The “peptide linkers” that link the non-affinity modified and/oraffinity modified IgSF domains can be a single amino acid residue orgreater in length. In some embodiments, the peptide linker has at leastone amino acid residue but is no more than 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues inlength. In some embodiments, the linker is (in one-letter amino acidcode): GGGGS (“4GS”) or multimers of the 4GS linker, such as repeats of2, 3, 4, or 5 4GS linkers. In further optional embodiments, a series ofalanine residues are interposed between 4GS linkers and to an Fc towhich the immunomodulatory protein is covalently linked. In someembodiments, the number of alanine residues in each series is: 2, 3, 4,5, or 6 alanines.

A. Exemplary Affinity Modified IgSF Domains

In some embodiments, the affinity modified IgSF domain has one or moreamino acid substitutions in an IgSF domain of a wild-type or unmodifiedIgSF protein, such as set forth in Table 1 above. The one or more aminoacid substitutions can be in the ectodomain of the wild-type orunmodified IgSF domain, such as the extracellular domain. In someembodiments, the one or more amino acid substitutions are in the IgVdomain or specific binding fragment thereof. In some embodiments, theone or more amino acid substitutions are in the IgC domain or specificbinding fragment thereof. In some embodiments of the affinity modifiedIgSF domain, some of the one or more amino acid substitutions are in theIgV domain or a specific binding fragment thereof, and some of the oneor more amino acid substitutions are in the IgC domain or a specificbinding fragment thereof.

In some embodiments, the affinity modified IgSF domain has up to 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacid substitutions. The substitutions can be in the IgV domain or theIgC domain. In some embodiments, the affinity modified IgSF domain hasup to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 amino acid substitutions in the IgV domain or specific bindingfragment thereof. In some embodiments, the affinity modified IgSF domainhas up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acid substitutions in the IgC domain or specific bindingfragment thereof. In some embodiments, the affinity modified IgSF domainhas at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity with the wild-type orunmodified IgSF domain or specific binding fragment thereof, such as theIgSF domain contained in the IgSF protein set forth in any of SEQ IDNOS: 1-27.

In some embodiments, the affinity modified IgSF domain contains one ormore amino acid substitutions in a wild-type or unmodified IgSF domainof a B7 IgSF family member. In some embodiments, the B7 IgSF familymember is CD80, CD86 or ICOS Ligand (ICOSL). In some embodiments, theaffinity modified IgSF domain has at least about 85%, 86%, 86%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with the wild-type or unmodified IgSF domain or specificbinding fragment thereof, such as the IgSF domain contained in the IgSFprotein set forth in any of SEQ ID NOS: 1, 2 or 5. Exemplary affinitymodified IgSF domains of CD80 are set forth in Table 2. Exemplaryaffinity modified IgSF domains of ICOSL are set forth in Table 3.Exemplary affinity modified IgSF domains of CD86 are set forth in Table4.

TABLE 2 Exemplary variant CD80 polypeptides ECD IgV SEQ ID SEQ IDMutation(s) NO NO Wild-type 28 152 L70Q/A91G 55 153 L70Q/A91G/T130A 56L70Q/A91G/I118A/T120S/T130A 57 V4M/L70Q/A91G/T120S/T130A 58 154L70Q/A91G/T120S/T130A 59 V20L/L70Q/A91S/T120S/T130A 60 155S44P/L70Q/A91G/T130A 61 156 L70Q/A91G/E117G/T120S/T130A 62A91G/T120S/T130A 63 157 L70R/A91G/T120S/T130A 64 158L70Q/E81A/A91G/T120S/I127T/T130A 65 159 L70Q/Y87N/A91G/T130A 66 160T28S/L70Q/A91G/E95K/T120S/T130A 67 161 N63S/L70Q/A91G/T120S/T130A 68 162K36E/I67T/L70Q/A91G/T120S/T130A/N152T 69 163 E52G/L70Q/A91G/T120S/T130A70 164 K37E/F59S/L70Q/A91G/T120S/T130A 71 165 A91G/S103P 72 K89E/T130A73 166 A91G 74 D60V/A91G/T120S/T130A 75 167 K54M/A91G/T120S 76 168M38T/L70Q/E77G/A91G/T120S/T130A/N152T 77 169R29H/E52G/L70R/E88G/A91G/T130A 78 170 Y31H/T41G/L70Q/A91G/T120S/T130A 79171 V68A/110A 80 172 S66H/D90G/T110A/F116L 81 173 R29H/E52G/T120S/T130A82 174 A91G/L102S 83 I67T/L70Q/A91G/T120S 84 175L70Q/A91G/T110A/T120S/T130A 85 M38V/T41D/M43I/W50G/D76G/V83A/K89E/ 86176 T120S/T130A V22A/L70Q/S121P 87 177A12V/S15F/Y31H/T41G/T130A/P137L/N152T 88 178I67F/L70R/E88G/A91G/T120S/T130A 89 179 E24G/L25P/L70Q/T120S 90 180A91G/F92L/F108L/T120S 91 181 R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/ 92 182M47T/E81V/L85R/K89N/A91T/F92P/K93V/ R94L/I118T/N149SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/ 93M47T/E81V/L85R/K89N/A91T/F92P/K93V/ R94L/N144S/N149SR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/ 94 183M43R/M47T/E81V/L85R/K89N/A91T/F92P/ K93V/R94L/L148S/N149SE24G/R29D/Y31L/Q33H/K36G/M38I/T41A/ 95 184M43R/M47T/F59L/E81V/L85R/K89N/A91T/ F92P/K93V/R94L/H96R/N149S/C182SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/ 96 M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S R29V/M43Q/E81R/L85I/K89R/D90L/ 97 185A91E/F92N/K93Q/R94G T41I/A91G 98 186K89R/D90K/A91G/F92Y/K93R/N122S/N177S 99 187 K89R/D90K/A91G/F92Y/K93R 100K36G/K37Q/M38I/F59L/E81V/L85R/ 101 188 K89N/A91T/F92P/K93V/R94L/E99G/T130A/N149S E88D/K89R/D90K/A91G/F92Y/K93R 102 189 K36G/K37Q/M38I/L40M103 190 K36G 104 191 R29H/Y31H/T41G/Y87N/E88G/K89E/ 105 192D90N/A91G/P109S A12T/H18L/N43V/F59L/E77K/P109S/I118T 106 193R29V/Y31F/K36G/M38L/N43Q/E81R/V83I/ 107 194L85I/K89R/D90L/A91E/F92N/K93Q/R94G V68M/L70P/L72P/K86E 108 195

TABLE 3 Exemplary variant ICOSL polypeptides ECD IgV SEQ ID SEQ IDMutation(s) NO NO Wild-type 32 196 N52S 109 197 N52H 110 198 N52D 111199 N52Y/N57Y/F138L/L203P 112 200 N52H/N57Y/Q100P 113 201N52S/Y146C/Y152C 114 N52H/C198R 115 N52H/C140D/T225A 116N52H/C198R/T225A 117 N52H/K92R 118 202 N52H/S99G 119 203 N52Y 120 204N57Y 121 205 N57Y/Q100P 122 206 N52S/S130G/Y152C 123 N52S/Y152C 124N52S/C198R 125 N52Y/N57Y/Y152C 126 N52Y/N57Y/129P/C198R 127N52H/L161P/C198R 128 N52S/T113E 129 S54A 130 207 N52D/S54P 131 208N52K/L208P 132 209 N52S/Y152H 133 N52D/V151A 134 N52H/I143T 135N52S/L80P 136 210 F120S/Y152H/N201S 137 N52S/R75Q/L203P 138 211N52S/D158G 139 N52D/Q133H 140 N52S/N57Y/H94D/L96F/L98F/Q100R 141 212N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S 142 213 N52S/G103E 239 240

TABLE 4 Exemplary variant CD86 polypeptides ECD IgC SEQ ID SEQ IDMutation(s) NO NO Wild-type 29 220 Q35H/H90L/Q102H 148 221 Q35H 149 222H90L 150 223 Q102H 151 224

In some embodiments, the affinity modified IgSF domain contains one ormore amino acid substitutions in a wild-type or unmodified IgSF domainof an NkP30 family member. In some embodiments, the affinity modifiedIgSF domain has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with thewild-type or unmodified IgSF domain or specific binding fragmentthereof, such as the IgSF domain contained in the IgSF protein set forthin SEQ ID NO:27. Table 5 provides exemplary affinity modified NkP30 IgSFdomains.

TABLE 5 Exemplary variant NKp30 polypeptides ECD IgV SEQ ID SEQ IDMutation(s) NO NO Wild-type 54 214 L30V/A60V/S64P/S86G 143 215 L30V 144216 A60V 145 217 S64P 146 218 S86G 147 219

B. Types of Affinity-Modified Immunomodulatory Protein

1. Dual Binding Affinity Modified Domain

In some embodiments, the immunomodulatory protein provided herein cancomprise the sequence of at least one IgSF domain of a wild-typemammalian non-immunoglobulin (i.e., non-antibody) IgSF family member,wherein at least one IgSF domain therein is affinity modified (“Type I”immunomodulatory proteins). In some embodiments, the at least onemodified IgSF domain specifically binds non-competitively to the atleast two cognate binding partners.

In some embodiments, the immunomodulatory protein comprises at least onenon-immunoglobulin affinity modified immunoglobulin superfamily (IgSF)domain that specifically binds non-competitively to at least two cognatebinding partners. In some embodiments, the affinity modified domainexhibits increased binding to at least one of the cognate bindingpartners compared to the wild-type or unmodified IgSF domain. In someembodiments, the affinity modified domain exhibits increased binding toat least two different cognate binding partners.

In some embodiments of a Type I immunomodulatory protein of theinvention, the unmodified or wild-type IgSF member, such as mammalianIgSF member, is one of the IgSF members or comprise an IgSF domain fromone of the IgSF members as indicated in Table 1 including any mammalianorthologs thereof.

In some embodiments, additional IgSF domains present within the Type Iimmunomodulatory protein can be non-affinity modified and/or affinitymodified, such as at least two, three, four, or five IgSF domains and,in some embodiments, exactly two, three, four, or five IgSF domains.

In some embodiments, a Type I immunomodulatory protein herein comprisingat least one non-immunoglobulin affinity modified immunoglobulinsuperfamily (IgSF) domain comprising one or more amino acidsubstitution(s) in a wild-type or unmodified IgSF domain, in which theaffinity modified IgSF domain has 1) altered, e.g. increased ordecreased, binding to at least two cognate binding partners compared tothe wild-type or unmodified IgSF domain; and 2) the at least oneaffinity modified IgSF domain specifically binds non-competitively tothe at least two cognate binding partners. In some embodiments, theaffinity modified IgSF domain of the Type I immunomodulatory protein hasincreased binding to at least two cognate binding partners. In someembodiments, the affinity modified IgSF domain of the Type Iimmunomodulatory protein has decreased binding to at least two cognatebinding partners. In some embodiments, the affinity modified IgSF domainof the Type I immunomodulatory protein has increased binding to at leastone cognate binding partner and decreased binding to at least one otherdifferent cognate binding partner.

In some embodiments, the two cognate binding partners are expressed onthe surface of at least two different cells, such as two differentmammalian cells. For example, in some embodiments, one cognate bindingpartner is expressed on a lymphocyte and another cognate binding partneris expressed on an antigen-presenting cells. In some embodiments, thetwo cognate binding partners are expressed on the same cell type, suchas same immune cell. In some embodiment, the Type I immunomodulatoryprotein is capable of modulating the immunological activity of one ormore of the immune cells, such as the immunological activity of alymphocyte, for example, a T cell. In some embodiments, immunologicalactivity is increased. In some embodiments, immunological activity isdecreased.

In some embodiments, the immunomodulatory protein comprises or consistsessentially of only one affinity modified IgSF domain, which bindsnon-competitively to the at least two cognate binding partners. In someembodiments, the affinity modified domain is an affinity modified IgVdomain. In some embodiments, the affinity modified domain is an affinitymodified IgC domain.

In some embodiments, a Type I immunomodulatory protein provided hereincomprises an affinity modified CD80 IgSF domain that non-competitivelyspecifically binds to CD28 and PDL1. In some embodiments, the affinitymodified CD80 IgSF domain is an IgV domain. In some embodiments, theimmunomodulatory protein further comprises a further non-affinitymodified IgSF domain.

2. Stacked or Multi-Domain Immunomodulatory Proteins

In some embodiments of the present invention, an immunomodulatoryprotein comprises a combination (a “non-wild-type combination”) and/orarrangement (a “non-wild type arrangement” or “non-wild-typepermutation”) of an affinity modified and/or non-affinity modified IgSFdomain sequences that are not found in wild-type IgSF family members(“Type II” immunomodulatory proteins). The sequences of the IgSF domainswhich are non-affinity modified (e.g., wild-type) or have been affinitymodified can be mammalian, such as from mouse, rat, cynomolgus monkey,or human origin, or combinations thereof. The number of suchnon-affinity modified or affinity modified IgSF domains present in theseembodiments of a Type II immunomodulatory protein (whether non-wild typecombinations or non-wild type arrangements) is at least 2, 3, 4, or 5and in some embodiments exactly 2, 3, 4, or 5 IgSF domains (wherebydetermination of the number of affinity modified IgSF domains disregardsany non-specific binding fractional sequences thereof and/orsubstantially immunologically inactive fractional sequences thereof).

In some embodiments, the Type II immunomodulatory proteins of theinvention comprise a non-wild type combination of IgSF domains whereinthe IgSF domains can be an IgSF domain of an IgSF family member fromthose listed in Table 1. Thus, in some embodiments, the immunodulatoryprotein can contain a first and second IgSF domain that can each be anaffinity-modified IgSF domain containing one or more amino acidsubstitutions compared to an IgSF domain contained in an IgSF familymember set forth in Table 1.

In some embodiments, IgSF domains are each independently an affinity ornon-affinity modified IgSF domain contained in an IgSF family member ofa family selected from Signal-Regulatory Protein (SIRP) Family,Triggering Receptor Expressed On Myeloid Cells Like (TREML) Family,Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) Family,Sialic Acid Binding Ig-Like Lectin (SIGLEC) Family, Butyrophilin Family,B7 family, CD28 family, V-set and Immunoglobulin Domain Containing(VSIG) family, V-set transmembrane Domain (VSTM) family, MajorHistocompatibility Complex (MHC) family, Signaling lymphocyticactivation molecule (SLAM) family, Leukocyte immunoglobulin-likereceptor (LIR), Nectin (Nec) family, Nectin-like (NECL) family,Poliovirus receptor related (PVR) family, Natural cytotoxicitytriggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM)family or Killer-cell immunoglobulin-like receptors (KIR) family. Insome embodiments, the IgSF domains are each independently derived froman IgSF protein selected from the group consisting of CD80(B7-1),CD86(B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2(PD-L2, CD273), ICOSLG(B7RP1,CD275, ICOSL, B7-H2), CD276(B7-H3), VTCN1(B7-H4), CD28, CTLA4,PDCD1(PD-1), ICOS, BTLA(CD272), CD4, CD8A(CD8-alpha), CD8B(CD8-beta),LAG3, HAVCR2(TIM-3), CEACAM1, TIGIT, PVR(CD155), PVRL2(CD112), CD226,CD2, CD160, CD200, CD200R1(CD200R), and NC R3 (NKp30).

In some embodiments, the IgSF domains independently contain one or moreamino acid substitutions compared to an IgSF domain in a wild-type orunmodified IgSF domain, such as an IgSF domain in an IgSF family memberset forth in Table 1. In some embodiments, the affinity-modified IgSFdomain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type orunmodified IgSF domain or a specific binding fragment thereof containedin the sequence of amino acids set forth in any of SEQ ID NOS: 1-27. Insome embodiments, the wild-type or unmodified IgSF domain is an IgVdomain or an IgC domain, such as an IgC1 or IgC2 domain. In someembodiments, the affinity modified IgSF domain is an affinity-modifiedIgV domain or IgC domain.

In some embodiments of a Type II immunomodulatory protein of theinvention, the number of IgSF domains is at least 2 wherein the numberof affinity modified and the number of non-affinity modified IgSFdomains is each independently at least: 0, 1, 2, 3, 4, 5, or 6. Thus,the number of affinity modified IgSF domains and the number ofnon-affinity modified IgSF domains, respectively, (affinity modifiedIgSF domain: non-affinity modified IgSF domain), can be exactly or atleast: 2:0 (affinity modified: wild-type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2,2:4, 4:2, 1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1.

In some embodiments of a Type II immunomodulatory protein, at least twoof the non-affinity modified and/or affinity modified IgSF domains areidentical IgSF domains.

In some embodiments, a Type II immunomodulatory protein of the presentinvention comprises at least two affinity modified and/or non-affinitymodified IgSF domains from a single IgSF member but in a non-wild-typearrangement (alternatively, “permutation”). One illustrative example ofa non-wild type arrangement or permutation is an immunomodulatoryprotein of the present invention comprising a non-wild-type order ofaffinity modified and/or non-affinity modified IgSF domain sequencesrelative to those found in the wild-type mammalian IgSF family memberwhose IgSF domain sequences served as the source of the non-affinitymodified and/or affinity modified IgSF domains. The mammalian wild-typeIgSF members in the preceding embodiment specifically includes thoselisted in Table 1. Thus, in one example, if the wild-type family membercomprises an IgC1 domain proximal to the transmembrane domain of a cellsurface protein and an IgV domain distal to the transmembrane domain,then an immunomodulatory protein of the present invention can comprisean IgV proximal and an IgC1 distal to the transmembrane domain albeit ina non-affinity modified and/or affinity modified form. The presence, inan immunomodulatory protein of the present invention, of bothnon-wild-type combinations and non-wild-type arrangements ofnon-affinity modified and/or affinity modified IgSF domains is alsowithin the scope of the present invention.

In some embodiments of a Type II immunomodulatory protein, thenon-affinity modified and/or affinity modified IgSF domains arenon-identical (i.e., different) IgSF domains. Non-identical affinitymodified IgSF domains specifically bind, under specific bindingconditions, different cognate binding partners and are “non-identical”irrespective of whether or not the wild-type IgSF domains from whichthey are engineered was the same. Thus, for example, a non-wild-typecombination of at least two non-identical IgSF domains in animmunomodulatory protein of the present invention can comprise at leastone IgSF domain sequence whose origin is from and unique to one IgSFfamily member, and at least one of a second IgSF domain sequence whoseorigin is from and unique to another IgSF family member, wherein theIgSF domains of the immunomodulatory protein are in non-affinitymodified and/or affinity modified form. However, in alternativeembodiments, the two non-identical IgSF domains originate from the sameIgSF domain sequence but at least one is affinity modified such thatthey specifically bind to different cognate binding partners.

In some embodiments, the number of non-identical non-affinity modifiedand/or affinity modified IgSF domains present in an immunomodulatoryprotein of the invention is at least 2, 3, 4, or 5 and in someembodiments exactly 2, 3, 4, or 5 non-identical non-affinity modifiedand/or affinity modified IgSF domains. In some embodiments, thenon-identical IgSF domains are combinations from at least two IgSFmembers indicated in Table 1, and in some embodiments at least 3 or 4IgSF members of Table 1.

In some specific embodiments, a Type II immunomodulatory protein of theinvention comprises an affinity modified NKp30 IgSF domain and anaffinity modified ICOSLG IgSF domain, affinity modified CD80 IgSF domainor an affinity modified CD86 IgSF domain. In some embodiments, a Type IIimmunomodulatory protein comprises an affinity modified IgSF domain fromat least two B7 family members. In some embodiments, theimmunomodulatory proteins comprises at least two affinity modifieddomains from an affinity modified CD80 IgSF domain, an affinity modifiedICOSL IgSF domain or an affinity modified CD86 IgSF domain or specificbinding fragments thereof. In some embodiments, the affinity modifieddomains are linked via at least or exactly 1, 2, 3, 4 G4S domains.

A plurality of non-affinity modified and/or affinity modified IgSFdomains in a stacked immunomodulatory protein polypeptide chain need notbe covalently linked directly to one another. In some embodiments, anintervening span of one or more amino acid residues indirectlycovalently bonds the non-affinity modified and/or affinity modified IgSFdomains to each other. The linkage can be via the N-terminal toC-terminal residues.

In some embodiments, the linkage can be made via side chains of aminoacid residues that are not located at the N-terminus or C-terminus ofthe non-affinity modified and/or affinity modified IgSF domain. Thus,linkages can be made via terminal or internal amino acid residues orcombinations thereof.

In some embodiments, the “peptide linkers” that link the non-affinitymodified and/or affinity modified IgSF domains can be a single aminoacid residue or greater in length. In some embodiments, the peptidelinker has at least one amino acid residue but is no more than 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid residues in length. In some embodiments, the linker is (inone-letter amino acid code): GGGGS (“4GS”) or multimers of the 4GSlinker, such as repeats of 2, 3, 4, or 5 4GS linkers. In furtheroptional embodiments, a series of alanine residues are interposedbetween a peptide linker (such as a 4GS linker or multimer thereof) andan Fc to which the immunomodulatory protein is covalently linked. Insome embodiments, the number of alanine residues in each series is: 2,3, 4, 5, or 6 alanines.

In some embodiments, the non-affinity modified and/or affinity modifiedIgSF domains are linked by “wild-type peptide linkers” inserted at theN-terminus and/or C-terminus of the first and/or second non-affinitymodified and/or affinity modified IgSF domains. In some embodiments,there is present a leading peptide linker inserted at the N-terminus ofthe first IgSF domain and/or a first trailing sequence inserted at theC-terminus of the first non-affinity modified and/or affinity modifiedIgSF domain. In some embodiments, there is present a second leadingpeptide linker inserted at the N-terminus of the second IgSF domainand/or a second trailing sequence inserted at the C-terminus of thesecond non-affinity modified and/or affinity modified IgSF domain. Whenthe first and second non-affinity modified and/or affinity modified IgSFdomains are derived from the same parental protein and are connected inthe same orientation, wild-type peptide linkers between the first andsecond non-affinity modified and/or affinity modified IgSF domains arenot duplicated. For example, when the first trailing wild-type peptidelinker and the second leading wild-type peptide linker are the same, theType II immunomodulatory protein does not comprise either the firsttrailing wild-type peptide linker or the second leading wild-typepeptide linker.

In some embodiments, the Type II immunomodulatory protein comprises afirst leading wild-type peptide linker inserted at the N-terminus of thefirst non-affinity modified and/or affinity modified IgSF domain,wherein the first leading wild-type peptide linker comprises at least 5(such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore) consecutive amino acids from the intervening sequence in thewild-type protein from which the first non-affinity modified and/oraffinity modified IgSF domain is derived between the parental IgSFdomain and the immediately preceding domain (such as a signal peptide oran IgSF domain). In some embodiments, the first leading wild-typepeptide linker comprises the entire intervening sequence in thewild-type protein from which the first non-affinity modified and/oraffinity modified IgSF domain is derived between the parental IgSFdomain and the immediately preceding domain (such as a signal peptide oran IgSF domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a first trailing wild-type peptide linker inserted at theC-terminus of the first non-affinity modified and/or affinity modifiedIgSF domain, wherein the first trailing wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the first non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain). In some embodiments, the firsttrailing wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the first non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a second leading wild-type peptide linker inserted at theN-terminus of the second non-affinity modified and/or affinity modifiedIgSF domain, wherein the second leading wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately preceding domain (such as asignal peptide or an IgSF domain). In some embodiments, the secondleading wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately preceding domain (such as asignal peptide or an IgSF domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a second trailing wild-type peptide linker inserted at theC-terminus of the second non-affinity modified and/or affinity modifiedIgSF domain, wherein the second trailing wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain). In some embodiments, the secondtrailing wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain).

Exemplary of a leading sequence and trailing sequence for a Type IIprotein containing a CD80 IgSF domain is set forth in SEQ ID NO:231 andSEQ ID NO:232. Exemplary of a leading sequence and trailing sequence fora Type II protein containing an ICOSL IgSF domain is set forth in SEQ IDNO: 233 and 234. Exemplary of a leading sequence and a trailing sequencefor a Type II protein containing an CD86 IgSF domain is set forth in anyof SEQ ID NOS: 236-238. Exemplary of a wild-type linker sequence for aType II protein containing an NKp30 IgSF domain is set forth in SEQ IDNO:235.

C. Format of Affinity-Modified Immunomodulatory Protein

In some embodiments an immunomodulatory protein provided herein is insoluble form. Those of skill will appreciate that cell surface proteinstypically have an intracellular, transmembrane, and extracellular domain(ECD) and that a soluble form of such proteins can be made using theextracellular domain or an immunologically active subsequence thereof.Thus, in some embodiments, the immunomodulatory protein containing anaffinity modified IgSF domain lacks a transmembrane domain or a portionof the transmembrane domain. In some embodiments, the immunomodulatoryprotein containing an affinity modified IgSF domain lacks theintracellular (cytoplasmic) domain or a portion of the intracellulardomain. In some embodiments, the immunomodulatory protein contains anaffinity modified IgSF domain that only contains the ECD domain or aportion thereof containing an IgV domain and/or IgC domain or specificbinding fragments thereof.

In some embodiments, the soluble form of an immunomodulatory protein ofthe present invention is covalently bonded, directly or indirectly, toan immunoglobulin Fc. Generally, the Fc is covalently bonded to theamino terminus of the immunomodulatory protein. The immunoglobulin Fc isin some embodiments a mammalian IgG class immunoglobulin, such as IgG1or IgG2. In particular embodiments, the Fc will be human IgG1 or IgG2Fc. It will be appreciated by those of skill in the art that smallchanges, such as 1, 2, 3, or 4, amino acid substitutions, deletions,additions, or combinations thereof, can be made to an Fc withoutsubstantially changing its pharmacokinetic properties. Such changes madebe made, for example, to aid in manufacturability or to enhance,suppress, or eliminate antibody-dependent cell-mediated cytotoxicity.The term “Fc” as used herein is meant to embrace such molecules.

In some embodiments, the Fc is murine or human Fc. In some embodiments,the Fc is derived from IgG1, such as human IgG1. In some embodiments,the Fc comprises the amino acid sequence set forth in SEQ ID NO: 226 ora sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to SEQ ID NO:226. In some embodiments, the Fc is derived fromIgG2, such as human IgG2. In some embodiments, the Fc comprises theamino acid sequence set forth in SEQ ID NO: 227 or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ IDNO:227.

In some embodiments, one or more amino acid modifications may beintroduced into the Fc region of an IgSF-Fc variant fusion providedherein, thereby generating an Fc region variant. In some embodiments,the Fc region variant has decreased effector function. There are manyexamples of changes or mutations to Fc sequences that can alter effectorfunction. For example, WO 00/42072, WO2006019447 and Shields et al. JBiol. Chem. 9(2): 6591-6604 (2001) describe exemplary Fc variants withimproved or diminished binding to FcRs. The contents of thosepublications are specifically incorporated herein by reference.

In some embodiments, the Fc region that possesses some but not alleffector functions, which makes it a desirable candidate forapplications in which the half-life of the Fc fusion in vivo isimportant yet certain effector functions (such as CDC and ADCC) areunnecessary or deleterious. In vitro and/or in vivo cytotoxicity assayscan be conducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the Fc-ICOSL variant fusion lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of invitro assays to assess ADCC activity of a molecule of interest isdescribed in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al.Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.,Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assay methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the Fc-ICOSL variant fusion isunable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3cbinding ELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Fc fusions with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 by EU numbering (U.S. Pat. No. 6,737,056). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327 by EU numbering, including theso-called “DANA” Fc mutant with substitution of residues 265 and 297 toalanine (U.S. Pat. No. 7,332,581).

Certain Fc variants with improved or diminished binding to FcRs aredescribed. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312,WO2006019447 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In some embodiments, alterations are made in the Fc region that resultin diminished C1q binding and/or Complement Dependent Cytotoxicity(CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, andIdusogie et al., J. Immunol. 164: 4178-4184 (2000).

In some embodiments, there is provided an ICOSL-Fc variant fusioncomprising a variant Fc region comprising one or more amino acidsubstitutions which increase half-life and/or improve binding to theneonatal Fc receptor (FcRn). Antibodies with increased half-lives andimproved binding to FcRn are described in US2005/0014934A1 (Hinton etal.). Those antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Such Fc variants include those with substitutions at one or more of Fcregion residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317,340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 by EU numbering,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

In some embodiments, the Fc is an IgG1 variant that contains at leastone amino acid substitution that is N82G by numbering of SEQ ID NO:226(corresponding to N297G by EU numbering). In some embodiments, thevariant Fc region further comprises a C5S amino acid modification. Forexample, in some embodiments, the variant Fc region comprises thefollowing amino acid modifications: C5S and N82G.

In some embodiments, indirect covalent bonding of an Fc to animmunomodulatory protein of the present invention can be made via, forexample, via a single amino acid or via a peptide (two or more aminoacid residues in length) linker. Further, the single polypeptide chainsof such Fc-fusion molecules can be dimerized through a variety of meansincluding via inter-polypeptide chain disulfide bonds. Dimerized formsof the immunomodulatory proteins of the invention can comprise twoidentical or substantially identical species of polypeptides of theinvention (homodimers), or separate species of polypeptide chains of theinvention (heterodimers). It will be appreciated thatmicroheterogeneities can exist even between the same species ofpolypeptide chain owing to minor differences in amino-terminus andcarboxy-terminus residues from minor differences in expression orproteolysis, or to differences resulting from post-translationalmodification. Nonetheless, such substantially identical chains aredeemed to be homodimeric. Derivatized immunomodulatory proteins arewithin the scope of the present invention and are often made to, forexample, provide altered physico-chemical or pharmacokinetic properties.

In even more specific embodiments, the preceding specific embodimentsare covalently linked to an Fc, such as a human IgG1 or IgG2 domain. Ina further specific embodiment, the Fc is attached to an immunomodulatoryprotein via one or more G4S domains, often with at least or exactly one,two, three, four, or five successive alanine residues directly linked tothe Fc and to the immunomodulatory protein.

In other embodiments, an immunomodulatory protein provided herein isbound to a liposomal membrane. A variety of methods to covalently ornon-covalently attach proteins to a liposome surface are known in theart such as by amide conjugation or disulfide/thioether conjugation.

D. Functional Activity of Immunomodulatory Proteins

In some embodiments, the immunomodulatory proteins containing anaffinity modified IgSF domain provided herein (full-length and/orspecific binding fragments or stack constructs or fusion thereof)exhibit immunomodulatory activity to modulate T cell activation.Functionally, and irrespective of whether specific binding to itscognate binding partner is increased or decreased, the immunomodulatoryproteins provided herein act to enhance or suppress immunologicalactivity of lymphocytes relative to lymphocytes under the appropriateassay controls, such as in an MLR assay. In some embodiments, animmunomodulatory protein provided herein comprises at least two affinitymodified IgSF domains wherein at least one of the affinity modified IgSFdomains acts to enhance immunological activity and at least one affinitymodified IgSF domain acts to suppress immunological activity.

In some embodiments, the provided immunomodulatory proteins modulateIFN-gamma expression in a primary T cell assay relative to a wild-typeor unmodified IgSF domain control. In some cases, modulation ofIFN-gamma expression can increase or decrease IFN-gamma expressionrelative to the control. Assays to determine specific binding andIFN-gamma expression are well-known in the art and include the MLR(mixed lymphocyte reaction) assays measuring interferon-gamma cytokinelevels in culture supernatants (Wang et al., Cancer Immunol Res. 2014September: 2(9):846-56), SEB (staphylococcal enterotoxin B) T cellstimulation assay (Wang et al., Cancer Immunol Res. 2014 September:2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander,J Transl Med. 2010: 8: 104).

In some embodiments, an immunomodulatory protein containing an affinitymodified domain can in some embodiments increase or, in alternativeembodiments, decrease IFN-gamma (interferon-gamma) expression in aprimary T-cell assay relative to a wild-type IgSF domain control. Insome embodiments of the provided polypeptides containing an affinitymodified IgSF domain, the polypeptide can increase IFN-gamma expressionand, in alternative embodiments, decrease IFN-gamma expression in aprimary T-cell assay relative to a wild-type ICOSL control. In someembodiments of the provided polypeptides containing multiple affinitymodified IgSF domains, the polypeptide can increase IFN-gamma expressionand, in alternative embodiments, decrease IFN-gamma expression in aprimary T-cell assay relative to a wild-type IgSF domain control.

Those of skill will recognize that the format of the primary T-cellassay used to determine an increase in IFN-gamma expression can differfrom that employed to assay for a decrease in IFN-gamma expression. Inassaying for the ability of an immunomodulatory protein to decreaseIFN-gamma expression in a primary T-cell assay, a Mixed LymphocyteReaction (MLR) assay can be used as described in Example 6. In somecases, a soluble form of immunomodulatory protein can be employed todetermine the ability of the affinity modified IgSF domain to antagonizeand thereby decrease the IFN-gamma expression in a MLR as likewisedescribed in Example 6.

Alternatively, in assaying for the ability of an immunomodulatoryprotein to increase IFN-gamma expression in a primary T-cell assay, aco-immobilization assay can be used as described in Example 6. In aco-immobilization assay, a TCR signal, provided in some embodiments byanti-CD3 antibody, is used in conjunction with a co-immobilizedimmunomodulatory protein containing an affinity modified IgSF domain todetermine the ability to increase IFN-gamma expression relative to anIgSF domain control. In some cases, a soluble form of animmunomodulatory protein that is multimerized to a degree to providemultivalent binding can be employed to determine the ability of theimmunomodulatory protein to agonize and thereby increase the IFN-gammaexpression in a MLR as likewise described in Example 6.

Use of proper controls is known to those of skill in the art, however,in the aforementioned embodiments, the control typically involves use ofthe unmodified IgSF domain, such as a wild-type of native IgSF isoformfrom the same mammalian species from which the IgSF domain was derivedor developed. Irrespective of whether the binding affinity to either oneor both of cognate binding partners is increased or decreased, aparticular immunomodulatory protein in some embodiments will increaseIFN-gamma expression and, in alternative embodiments, decrease IFN-gammaexpression in a primary T-cell assay relative to a wild-type IgSF domaincontrol.

In some embodiments, an immunomodulatory protein, e.g. containing anaffinity modified IgSF domain, increases IFN-gamma expression (i.e.,protein expression) relative to a wild-type or unmodified IgSF domaincontrol by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, orhigher. In other embodiments, an immunomodulatory protein, e.g.containing an affinity modified IgSF domain, decreases IFN-gammaexpression (i.e. protein expression) relative to a wild-type orunmodified IgSF domain control by at least: 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or higher. In some embodiments, an enhancement ofimmunological activity can be an increase of at least 10%, 20%, 30%,40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than a non-zerocontrol value such as in a MLR assay. Wang et al., Cancer Immunol Res.2014 September: 2(9):846-56. In some embodiments, suppression ofimmunological activity can be a decrease of at least 10%, and up to 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90%.

III. Nucleic Acids and Methods of Making Proteins

The present invention provides isolated or recombinant nucleic acidscollectively referred to as “nucleic acids of the invention” whichencode any of the various embodiments of the immunomodulatory proteins(Type I and Type II) of the invention. Nucleic acids of the invention,including all described below, are useful in recombinant production(e.g., expression) of polypeptides of the invention. The nucleic acidsof the invention can be in the form of RNA or in the form of DNA, andinclude mRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. Thenucleic acids of the invention are typically DNA molecules, and usuallydouble-stranded DNA molecules. However, single-stranded DNA,single-stranded RNA, double-stranded RNA, and hybrid DNA/RNA nucleicacids or combinations thereof comprising any of the nucleotide sequencesof the invention also are provided.

The present invention also relates to expression vectors and host cellsuseful in producing the immunomodulatory proteins of the presentinvention. The immunomodulatory proteins of the invention can be made intransformed host cells using recombinant DNA techniques. To do so, arecombinant DNA molecule encoding an immunomodulatory protein isprepared. Methods of preparing such DNA molecules are well known in theart. For instance, sequences coding for the peptides could be excisedfrom DNA using suitable restriction enzymes. Alternatively, the DNAmolecule could be synthesized using chemical synthesis techniques, suchas the phosphoramidite method. Also, a combination of these techniquescould be used. In some instances, a recombinant or synthetic nucleicacid may be generated through polymerase chain reaction (PCR).

The invention also includes expression vectors capable of expressing theimmunomodulatory proteins in an appropriate host cell under conditionssuited to expression of the immunomodulatory protein. A recombinantexpression vector comprises the DNA molecule that codes for theimmunomodulatory protein operatively linked to appropriate expressioncontrol sequences. Methods of affecting this operative linking, eitherbefore or after the DNA molecule is inserted into the vector, are wellknown. Expression control sequences include promoters, activators,enhancers, operators, ribosomal binding sites, start signals, stopsignals, cap signals, polyadenylation signals, and other signalsinvolved with the control of transcription or translation. The resultingrecombinant expression vector having the DNA molecule thereon is used totransform an appropriate host. This transformation can be performedusing methods well known in the art. In some embodiments, a nucleic acidof the invention further comprises nucleotide sequence that encodes asecretory or signal peptide operably linked to the nucleic acid encodingan immunomodulatory protein of the invention such that theimmunomodulatory protein is recovered from the culture medium, hostcell, or host cell periplasm.

Any of a large number of available and well-known host cells can be usedin the practice of this invention. The selection of a suitable host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts can be equally effective for theexpression of a particular DNA sequence. Host cells can be a variety ofeukaryotic cells, such as in yeast cells, or with mammalian cells suchas Chinese hamster ovary (CHO) or HEK293 cells. Host cells can also beprokaryotic cells, such as with E. coli. The transformed host iscultured under immunomodulatory protein expressing conditions, and thenpurified. Recombinant host cells can be cultured under conventionalfermentation conditions so that the desired immunomodulatory proteinsare expressed. Such fermentation conditions are well known in the art.Finally, the immunomodulatory proteins are recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, and affinitychromatography. Protein refolding steps can be used, as desired, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed in the finalpurification steps. Immunomodulatory proteins of the present inventioncan also be made by synthetic methods. Solid phase synthesis is thepreferred technique of making individual peptides since it is the mostcost-effective method of making small peptides. For example, well knownsolid phase synthesis techniques include the use of protecting groups,linkers, and solid phase supports, as well as specific protection anddeprotection reaction conditions, linker cleavage conditions, use ofscavengers, and other aspects of solid phase peptide synthesis. Peptidescan then be assembled into the immunomodulatory proteins of the presentinvention.

The means by which the affinity modified IgSF domains of theimmunomodulatory invention are designed or created is not limited to anyparticular method. In some embodiments, however, wild-type IgSF domainsare mutagenized (site specific, random, or combinations thereof) fromwild-type IgSF genetic material and screened for altered bindingaccording to the methods disclosed in the Examples. Methods ormutagenizing nucleic acids is known to those of skill in the art. Insome embodiments, the affinity modified IgSF domains are synthesized denovo utilizing protein or nucleic acid sequences available at any numberof publicly available databases and then subsequently screened. TheNational Center for Biotechnology Information provides such informationand its website is publicly accessible via the internet as is theUniProtKB database as discussed previously.

IV. Methods of Screening or Identifying Affinity Modified IgSF Domains

Provided herein is a method of identifying an affinity modifiedimmunomodulatory protein that is capable of binding to two or morecognate binding partners at the same time or in a non-competitivemanner. In some embodiments, the method comprises: a) contacting amodified protein comprising at least one non-immunoglobulin modifiedimmunoglobulin superfamily (IgSF) domain or specific binding fragmentthereof with at least two cognate binding partners under conditionscapable of effecting binding of the protein with the at least twocognate binding partners, wherein the at least one modified IgSF domaincomprises one or more amino acid substitutions in a wild-type IgSFdomain; b) identifying a modified protein comprising the modified IgSFdomain that has increased binding to at least one of the two cognatebinding partners compared to a protein comprising the wild-type IgSFdomain; and c) selecting a modified protein comprising the modified IgSFdomain that binds non-competitively to the at least two cognate bindingpartners, thereby identifying the affinity modified immunomodulatoryprotein. In some embodiments, the affinity modified protein that isselected is capable or binding the two cognate binding partnerssimultaneously at the same time. It is within the level of a skilledartisan to assess or determine the presence of non-competitive bindinginteractions of a protein for two more different ligands. Exemplary ofsuch methods are described in Example 7.

In some embodiments, the IgSF domain is a non-immunoglobulin IgSFdomain. In some embodiments, the modified or variant protein is one inwhich one or more amino acid substitutions, deletions or insertions havebeen made in the IgSF domain of any of a non-immunoglobulin IgSF familymember, such as any set forth in Table 1. In some embodiments, themodified or variant IgSF domain or the modified protein containing themodified or variant IgSF domain contains at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes,such as amino acid substitutions.

In some embodiments, libraries of modified or variant proteins aregenerated by mutating any one or more amino acid residues of a proteinknown to contain a non-immunoglobulin IgSF domain using any methodcommonly known in the art. Any of the methods employed in the art forgenerating, engineering or diversifying a binding molecule can be usedin generating a modified IgSF domain herein. Exemplary of such methodsfor generating, engineering or diversifying a binding molecule include,but are not limited to, methods described in U.S. Pat. Nos. 5,223,409;5,571,698, 5,750,373, 5,821,047; 5,837,500; 5,733,743; 5,871,907;5,969,108; 6,040,136; 6,172,197; 6,291,159; 6,955,877; 6,979,538;6,831,161; 7,063,943; 7,118,879; 7,208,293; 7,332,571; 7,385,028;7,696,312; 7,638,299; 7,888,533; 7,642,044; U.S. Patent Application No.US20080300163; US20090208454; US20090155843; US20080113412;US20100035812; US20100093608; US20110015345; For example, anIgSF-contain can be used in methods in place of other binding moleculesin methods of diversification or engineering of biomolecules.

Methods for generating libraries of binding molecules and for creatingdiversity in the library are well known in the art and can be employedto generate libraries of protein variants. Approaches for generatingdiversity include targeted and non-targeted approaches well known in theart. For example, known approaches for generating diverse nucleic acidand polypeptide libraries include, but are not limited to error-pronePCR, cassette mutagenesis; mutual primer extension; template-assistedligation and extension; codon cassette mutagenesis;oligonucleotide-directed mutagenesis; amplification using degenerateoligonucleotide primers, including overlap and two-step PCR; andcombined approaches, such as combinatorial multiple cassette mutagenesis(CMCM) and related techniques. A skilled artisan is familiar with thesetechniques.

Examples of methods to mutate a protein to generate libraries ofcandidate modified protein molecules include methods that result inrandom mutagenesis across the entire protein sequence or methods thatresult in mutagenesis of a select region or domain of the protein.Mutations can be introduced randomly, using methods that result inrandom mutagenesis of the protein, or more systematically, using methodsthat specifically create single or multiple amino acid change at atargeted position. Both random mutagenesis and systematic site-directedmutagenesis can be used to introduce one or more mutations in theprotein. The variant protein can harbor one or more amino acidsubstitutions, insertions or deletions compared to the wildtype orunmodified protein used as the scaffold to generate the library. Thesubstitutions or insertions can be with any naturally occurring aminoacids or non-naturally occurring amino acids.

In methods for identifying or generating a variant or modified proteincontaining a non-immunoglobulin IgSF domain in accord with the providedmethods, one or more regions of the protein, such as an IgSF domain ordomains of the protein, can be modified using random mutagenesis of aregion to generate one or a plurality of modified protein molecules. Forexample, a library of variants can be generated containing a pluralityof modified molecules that each differ by at least one amino acidreplacement (i.e. substitution) deletion or insertion in an IgSF domaincompared to a corresponding unmodified or wild-type protein containingthe IgSF domain. The amino acid substitution(s) can be substitutions(s)of naturally occurring amino acids or non-naturally occurring aminoacids compared to the unmodified or wild-type protein. Generally, thelibraries provided herein include libraries containing at least 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 10², 10³, 10⁴, 2×10⁴, 3×10⁴, 4×10⁴,5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or moredifferent members. The generated library containing a plurality ofmodified proteins can be generated as a display library, including acombinatorial library where display of the variant is by, for example,phage display, cell-surface display, bead display, ribosome display, orothers. The libraries can be used to screen for modified or variantproteins containing non-immunoglobulin IgSF domains that specificallybind non-competitively to the at least two cognate binding partners.

In some embodiments, prior to selecting a modified protein comprisingthe modified IgSF domain that binds non-competitively to the at leasttwo cognate binding partners, the method includes combining two or moremodified IgSF domain or specific binding fragments thereof identified instep (b) to generate a stacked molecule construct containing a pluralityof different modified IgSF domains.

Thus, in some embodiments, provided herein is a method of identifying anaffinity modified immunomodulatory protein, comprising: a) contacting amodified protein comprising at least one non-immunoglobulin modifiedimmunoglobulin superfamily (IgSF) domain or specific binding fragmentthereof with at least two cognate binding partners under conditionscapable of effecting binding of the protein with the at least twocognate binding partners, wherein the at least one modified IgSF domaincomprises one or more amino acid substitutions in a wild-type IgSFdomain; b) identifying a modified protein comprising the modified IgSFdomain that has increased binding to at least one of the two cognatebinding partners compared to a protein comprising the wild-type IgSFdomain; c) combining two or more modified IgSF domains present in two ormore identified proteins to generate a fusion (stacked) proteincomprising a first modified IgSF domain linked to a second IgSF domain;and d) selecting a modified protein comprising the modified IgSF domainsthat binds non-competitively to the at least two cognate bindingpartners, thereby identifying the affinity modified immunomodulatoryprotein.

In some embodiments, the at least two cognate binding partners are cellsurface molecular species expressed on the surface of a mammalian cell.In some embodiments, the cell surface molecular species are expressed incis configuration or trans configuration. In some embodiments, themammalian cell is one of two mammalian cells forming an immunologicalsynapse (IS) and each of the cell surface molecular species is expressedon at least one of the two mammalian cells forming the IS. In someembodiments, at least one of the mammalian cells is a lymphocyte, whichcan be an NK cell or a T cell. In some embodiments, at least one of themammalian cells is a tumor cell. In some embodiments, at least one ofthe mammalian cells is an antigen-presenting cell.

In some embodiments, the two or more cognate binding partners areindependently a ligand of an IgSF member selected from CD80, CD86,PD-L1, PD-L2, ICOS Ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA,CD4, CD8-alpha, CD8-beta, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2,CD226, CD2, CD160, CD200, CD200R or Nkp30. In some embodiments, the twoor more cognate binding partners are independently a ligand of a B7family member. In some embodiments, the two or more cognate bindingpartners are selected from two or more of CD28, CTLA-4, ICOS or PD-L1.

V. Pharmaceutical Compositions and Formulations

A pharmaceutical composition comprising a therapeutic composition of theinvention may contain formulation materials for modifying, maintainingor preserving, for example, the pH, osmolarity, viscosity, clarity,color, isotonicity, odor, sterility, stability, rate of dissolution orrelease, adsorption, or penetration of the composition. The primaryvehicle or carrier in a pharmaceutical composition may be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier maybe water for injection or physiological saline, possibly supplementedwith other materials common in compositions for parenteraladministration. Neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. Other exemplary pharmaceuticalcompositions comprise Tris buffer of about pH 7.0-8.5, or acetate bufferof about pH 4.0-5.5, which may further include sorbitol or a suitablesubstitute therefore. In one embodiment of the present invention,binding agent compositions may be prepared for storage by mixing theselected composition having the desired degree of purity with optionalformulation agents in the form of a lyophilized cake or an aqueoussolution. Further, the binding agent product may be formulated as alyophilizate using appropriate excipients such as sucrose.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8. A particularlysuitable vehicle for parenteral administration is sterile distilledwater in which a binding agent is formulated as a sterile, isotonicsolution, properly preserved. Yet another preparation can involve theformulation of the desired molecule with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (polylacticacid, polyglycolic acid), beads, or liposomes, that provide for thecontrolled or sustained release of the product which may then bedelivered via a depot injection.

In another aspect, pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additional pharmaceutical compositions will be evident to thoseskilled in the art, including formulations involving binding agentmolecules in sustained- or controlled-delivery formulations. Techniquesfor formulating a variety of other sustained- or controlled-deliverymeans, such as liposome carriers, bio-erodible microparticles or porousbeads and depot injections, are also known to those skilled in the art.The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

In some embodiments, the pharmaceutical composition is sterile.Sterilization may be accomplished by filtration through sterilefiltration membranes or radiation. Where the composition is lyophilized,sterilization using this method may be conducted either prior to orfollowing lyophilization and reconstitution. The composition forparenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration. An effective amount of apharmaceutical composition to be employed therapeutically will depend,for example, upon the therapeutic context and objectives. One skilled inthe art will appreciate that the appropriate dosage levels for treatmentwill thus vary depending, in part, upon the molecule delivered, theindication for which the binding agent molecule is being used, the routeof administration, and the size (body weight, body surface or organsize) and condition (the age and general health) of the patient.Accordingly, the clinician may titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. The therapeuticcomposition of the invention can be administered parentally,subcutaneously, or intravenously, or as described elsewhere herein. Thetherapeutic composition of the invention may be administered in atherapeutically effective amount one, two, three or four times permonth, two times per week, biweekly (every two weeks), or bimonthly(every two months). Administration may last for a period of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 months or longer (e.g., one, two, three,four or more years, including for the life of the subject).

Generally, dosages and routes of administration of the pharmaceuticalcomposition are determined according to the size and condition of thesubject, according to standard pharmaceutical practice. For example, thetherapeutically effective dose can be estimated initially either in cellculture assays or in animal models such as mice, rats, rabbits, dogs,pigs, or monkeys. An animal model may also be used to determine theappropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage will be determined in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activecompound or to maintain the desired effect. Factors that may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy.

In some embodiments, the pharmaceutical composition is administered to asubject through any route, including orally, transdermally, byinhalation, intravenously, intra-arterially, intramuscularly, directapplication to a wound site, application to a surgical site,intraperitoneally, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intrapleurally, intraventricularly,intra-articularly, intraocularly, or intraspinally.

In some embodiments, the dosage of the pharmaceutical composition is asingle dose or a repeated dose. In some embodiments, the doses are givento a subject once per day, twice per day, three times per day, or fouror more times per day. In some embodiments, about 1 or more (such asabout 2 or more, about 3 or more, about 4 or more, about 5 or more,about 6 or more, or about 7 or more) doses are given in a week. In someembodiments, multiple doses are given over the course of days, weeks,months, or years. In some embodiments, a course of treatment is about 1or more doses (such as about 2 or more does, about 3 or more doses,about 4 or more doses, about 5 or more doses, about 7 or more doses,about 10 or more doses, about 15 or more doses, about 25 or more doses,about 40 or more doses, about 50 or more doses, or about 100 or moredoses).

In some embodiments, an administered dose of the pharmaceuticalcomposition is about 1 μg of protein per kg subject body mass or more(such as about 2 μg of protein per kg subject body mass or more, about 5μg of protein per kg subject body mass or more, about 10 μg of proteinper kg subject body mass or more, about 25 μg of protein per kg subjectbody mass or more, about 50 μg of protein per kg subject body mass ormore, about 100 μg of protein per kg subject body mass or more, about250 μg of protein per kg subject body mass or more, about 500 μg ofprotein per kg subject body mass or more, about 1 mg of protein per kgsubject body mass or more, about 2 mg of protein per kg subject bodymass or more, or about 5 mg of protein per kg subject body mass ormore).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. The exact dosage will bedetermined in light of factors related to the subject requiringtreatment. Dosage and administration are adjusted to provide sufficientlevels of the active compound or to maintain the desired effect. Factorsthat may be taken into account include the severity of the diseasestate, the general health of the subject, the age, weight, and gender ofthe subject, time and frequency of administration, drug combination(s),reaction sensitivities, and response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or biweekly depending on the half-life and clearance rate of theparticular formulation. The frequency of dosing will depend upon thepharmacokinetic parameters of the molecule in the formulation used.Typically, a composition is administered until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, or as multiple doses (at the same ordifferent concentrations/dosages) over time, or as a continuousinfusion. Further refinement of the appropriate dosage is routinelymade. Appropriate dosages may be ascertained through use of appropriatedose-response data.

In some embodiments, one or more biomarkers or physiological markers fortherapeutic effect can be monitored including T cell activation orproliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of various activation markers (e.g.,CD25, IL-2 receptor), inflammation, joint swelling or tenderness, serumlevel of C-reactive protein, anti-collagen antibody production, and/or Tcell-dependent antibody response(s).

An injectable pharmaceutical composition comprising a suitablepharmaceutically acceptable excipient or carrier (e.g., PBS) and aneffective amount of a therapeutic composition of the invention can beadministered parenterally, intramuscularly, intraperitoneally,intravenously, subdermally, transdermally, subcutaneously, orintradermally to a mammalian patient. Administration can be facilitatedvia liposomes. The skin and muscle are generally preferred targets foradministration of the therapeutic composition of the invention, by anysuitable technique. Thus, the delivery of the therapeutic composition ofthe invention into or through the skin of a mammalian subject (e.g.,human), is a feature of the invention. Such molecules of the inventioncan be administered in a pharmaceutically acceptable injectable solutioninto or through the skin, e.g., intramuscularly, or intraperitoneally.Administration can also be accomplished by transdermal devices, or, moretypically, biolistic delivery of the therapeutic composition of theinvention into, or through the skin of the subject or into exposedmuscle of the mammalian subject.

A variety of means are known for determining if administration of atherapeutic composition of the invention sufficiently modulatesimmunological activity by eliminating, sequestering, or inactivatingimmune cells mediating or capable of mediating an undesired immuneresponse; inducing, generating, or turning on immune cells that mediateor are capable of mediating a protective immune response; changing thephysical or functional properties of immune cells; or a combination ofthese effects. Examples of measurements of the modulation ofimmunological activity include, but are not limited to, examination ofthe presence or absence of immune cell populations (using flowcytometry, immunohistochemistry, histology, electron microscopy,polymerase chain reaction (PCR)); measurement of the functional capacityof immune cells including ability or resistance to proliferate or dividein response to a signal (such as using T cell proliferation assays andpepscan analysis based on 3H-thymidine incorporation followingstimulation with anti-CD3 antibody, anti-T cell receptor antibody,anti-CD28 antibody, calcium ionophores, PMA, antigen presenting cellsloaded with a peptide or protein antigen; B cell proliferation assays);measurement of the ability to kill or lyse other cells (such ascytotoxic T cell assays); measurements of the cytokines, chemokines,cell surface molecules, antibodies and other products of the cells(e.g., by flow cytometry, enzyme-linked immunosorbent assays, Westernblot analysis, protein microarray analysis, immunoprecipitationanalysis); measurement of biochemical markers of activation of immunecells or signaling pathways within immune cells (e.g., Western blot andimmunoprecipitation analysis of tyrosine, serine or threoninephosphorylation, polypeptide cleavage, and formation or dissociation ofprotein complexes; protein array analysis; DNA transcriptional,profiling using DNA arrays or subtractive hybridization); measurementsof cell death by apoptosis, necrosis, or other mechanisms (e.g., annexinV staining, TUNEL assays, gel electrophoresis to measure DNA laddering,histology; fluorogenic caspase assays, Western blot analysis of caspasesubstrates); measurement of the genes, proteins, and other moleculesproduced by immune cells (e.g., Northern blot analysis, polymerase chainreaction, DNA microarrays, protein microarrays, 2-dimensional gelelectrophoresis, Western blot analysis, enzyme linked immunosorbentassays, flow cytometry); and measurement of clinical symptoms oroutcomes such as improvement of autoimmune, neurodegenerative, and otherdiseases involving self proteins or self polypeptides (clinical scores,requirements for use of additional therapies, functional status, imagingstudies) for example, by measuring relapse rate or disease severity(using clinical scores known to the ordinarily skilled artisan) in thecase of multiple sclerosis, measuring blood glucose in the case of typeI diabetes, or joint inflammation in the case of rheumatoid arthritis.

Also provided herein are articles of manufacture comprising thepharmaceutical compositions described herein in suitable packaging.Suitable packaging for compositions (such as ophthalmic compositions)described herein are known in the art, and include, for example, vials(such as sealed vials), vessels, ampules, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Thesearticles of manufacture may further be sterilized and/or sealed.

Further provided are kits comprising the pharmaceutical compositions (orarticles of manufacture) described herein, which may further compriseinstruction(s) on methods of using the composition, such as usesdescribed herein. The kits described herein may also include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for performing any methods described herein.

VI. Therapeutic Applications

Immunomodulatory proteins of the invention are believed to have utilityin a variety of applications, including, but not limited to, e.g., inprophylactic or therapeutic methods (collectively, a “therapeuticcomposition of the invention”) for treating a variety of immune systemdiseases or conditions in a mammal in which modulation or regulation ofthe immune system and immune system responses is beneficial. Forexample, suppressing an immune response can be beneficial inprophylactic and/or therapeutic methods for inhibiting rejection of atissue, cell, or organ transplant from a donor by a recipient. In atherapeutic context, the mammalian subject is typically one with animmune system disease or condition, and administration is conducted toprevent further progression of the disease or condition. For example,administration of a therapeutic composition of the invention to asubject suffering from an immune system disease (e.g., autoimmunedisease) can result in suppression or inhibition of such immune systemattack or biological responses associated therewith. By suppressing thisimmune system attack on healthy body tissues, the resulting physicalsymptoms (e.g., pain, joint inflammation, joint swelling or tenderness)resulting from or associated with such attack on healthy tissues can bedecreased or alleviated, and the biological and physical damageresulting from or associated with the immune system attack can bedecreased, retarded, or stopped. In a prophylactic context, the subjectmay be one with, susceptible to, or believed to present an immune systemdisease, disorder or condition, and administration is typicallyconducted to prevent progression of the disease, disorder or condition,inhibit or alleviate symptoms, signs, or biological responses associatedtherewith, prevent bodily damage potentially resulting therefrom, and/ormaintain or improve the subject's physical functioning.

The immune system disease or disorder of the patient may be or involve,e.g., but is not limited to, Addison's Disease, Allergy, AlopeciaAreata, Alzheimer's, Antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis, Ankylosing Spondylitis, AntiphospholipidSyndrome (Hughes Syndrome), arthritis, Asthma, Atherosclerosis,Atherosclerotic plaque, autoimmune disease (e.g., lupus, RA, MS, Graves'disease, etc.), Autoimmune Hemolytic Anemia, Autoimmune Hepatitis,Autoimmune inner ear disease, Autoimmune Lymphoproliferative syndrome,Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Orchitis,Azoospermia, Behcet's Disease, Berger's Disease, Bullous Pemphigoid,Cardiomyopathy, Cardiovascular disease, Celiac Sprue/Coeliac disease,Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic idiopathicpolyneuritis, Chronic Inflammatory Demyelinating, Polyradicalneuropathy(CIPD), Chronic relapsing polyneuropathy (Guillain-Barré syndrome),Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold AgglutininDisease (CAD), COPD, CREST syndrome, Crohn's disease, Dermatitis,Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema,Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia,Evan's Syndrome, Exopthalmos, Fibromyalgia, Goodpasture's Syndrome,graft-related disease or disorder, Graves' Disease, GVHD, Hashimoto'sThyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic ThrombocytopeniaPurpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder(e.g., psoriasis), Inflammatory bowel disease (IBD), Insulin DependentDiabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes,Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki'sDisease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, lupus, LupusNephritis, Lymphoscytic Lypophisitis, Ménière's Disease, Miller FishSyndrome/acute disseminated encephalomyeloradiculopathy, MixedConnective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism,Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation,Pemphigus Foliaceus, Pemphigus Vulgaris, Pernicious Anaemia,Polyarteritis Nodosa, Polychondritis, Polyglandular Syndromes(Whitaker's syndrome), Polymyalgia Rheumatica, Polymyositis, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis/Autoimmune cholangiopathy,Psoriasis, Psoriatic arthritis, Raynaud's Phenomenon, Reiter'sSyndrome/Reactive arthritis, Restenosis, Rheumatic Fever, rheumaticdisease, Rheumatoid Arthritis, Sarcoidosis, Schmidt's syndrome,Scleroderma, Sjörgen's Syndrome, Solid-organ transplant rejection(kidney, heart, liver, lung, etc.), Stiff-Man Syndrome, Systemic LupusErythematosus (SLE), systemic scleroderma, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2diabetes, Ulcerative colitis, Uveitis, Vasculitis, Vitiligo, Wegener'sGranulomatosis, and preventing or suppressing an immune responseassociated with rejection of a donor tissue, cell, graft, or organtransplant by a recipient subject. Graft-related diseases or disordersinclude graft versus host disease (GVDH), such as associated with bonemarrow transplantation, and immune disorders resulting from orassociated with rejection of organ, tissue, or cell grafttransplantation (e.g., tissue or cell allografts or xenografts),including, e.g., grafts of skin, muscle, neurons, islets, organs,parenchymal cells of the liver, etc. With regard to a donor tissue,cell, graft or solid organ transplant in a recipient subject, it isbelieved that a therapeutic composition of the invention disclosedherein may be effective in preventing acute rejection of such transplantin the recipient and/or for long-term maintenance therapy to preventrejection of such transplant in the recipient (e.g., inhibitingrejection of insulin-producing islet cell transplant from a donor in thesubject recipient suffering from diabetes).

A therapeutic composition of the invention can also be used to inhibitgrowth of mammalian, particularly human, cancer cells as a monotherapy(i.e., as a single agent), in combination with at least onechemotherapeutic agent (i.e., a combination therapy), in combinationwith a cancer vaccine, in combination with an immune checkpointinhibitor and/or in combination with radiation therapy. In some aspectsof the present disclosure, the immune checkpoint inhibitor is nivolumab,tremelimumab, pembrolizumab, ipilimumab, or the like. An effectiveamount of a therapeutic composition is administered to inhibit, halt, orreverse progression of cancers that are sensitive to modulation ofimmunological activity by immunomodulatory proteins of the presentinvention. Human cancer cells can be treated in vivo, or ex vivo. In exvivo treatment of a human patient, tissue or fluids containing cancercells are treated outside the body and then the tissue or fluids arereintroduced back into the patient. In some embodiments, the cancer istreated in a human patient in vivo by administration of the therapeuticcomposition into the patient. Thus, the present invention provides exvivo and in vivo methods to inhibit, halt, or reverse progression of thetumor, or otherwise result in a statistically significant increase inprogression-free survival (i.e., the length of time during and aftertreatment in which a patient is living with cancer that does not getworse), or overall survival (also called “survival rate;” i.e., thepercentage of people in a study or treatment group who are alive for acertain period of time after they were diagnosed with or treated forcancer) relative to treatment with a control. The cancers which can betreated by the methods of the invention include, but are not limited to,melanoma, bladder cancer, hematological malignancies (leukemia,lymphoma, myeloma), liver cancer, brain cancer, renal cancer, breastcancer, pancreatic cancer (adenocarcinoma), colorectal cancer, lungcancer (small cell lung cancer and non-small-cell lung cancer), spleencancer, cancer of the thymus or blood cells (i.e., leukemia), prostatecancer, testicular cancer, ovarian cancer, uterine cancer, gastriccarcinoma, or Ewing's sarcoma.

VII. Exemplary Embodiments

Among the provided embodiments are:

Embodiment 1

In some embodiments, there is provided an immunomodulatory protein,comprising at least one non-immunoglobulin affinity modifiedimmunoglobulin superfamily (IgSF) domain comprising one or more aminoacid substitution(s) in a wild-type IgSF domain, wherein: the at leastone affinity modified IgSF domain has increased binding to at least twocognate binding partners compared to the wild-type IgSF domain; and theat least one affinity modified IgSF domain specifically bindsnon-competitively to the at least two cognate binding partners.

Embodiment 2

In some further embodiments of embodiment 1, the at least two cognatebinding partners are cell surface molecular species expressed on thesurface of a mammalian cell.

Embodiment 3

In some further embodiments of embodiment 2, the cell surface molecularspecies are expressed in cis configuration or trans configuration.

Embodiment 4

In some further embodiments of embodiment 2 or embodiment 3, themammalian cell is one of two mammalian cells forming an immunologicalsynapse (IS) and each of the cell surface molecular species is expressedon at least one of the two mammalian cells forming the IS.

Embodiment 5

In some further embodiments of any one of embodiments 2-4, at least oneof the mammalian cells is a lymphocyte.

Embodiment 6

In some further embodiments of embodiment 5, the lymphocyte is an NKcell or a T cell.

Embodiment 7

In some further embodiments of any one of embodiments 5-6, binding ofthe affinity modified IgSF domain modulates immunological activity ofthe lymphocyte.

Embodiment 8

In some further embodiments of embodiment 7, the immunomodulatoryprotein is capable of effecting increased immunological activitycompared the wild-type protein comprising the wild-type IgSF domain.

Embodiment 9

In some further embodiments of embodiment 7, the immunomodulatoryprotein is capable of effecting decreased immunological activitycompared to the wild-type protein comprising the wild-type IgSF domain.

Embodiment 10

In some further embodiments of any one of embodiments 2-9, at least oneof the mammalian cells is a tumor cell.

Embodiment 11

In some further embodiments of any one of embodiments 2-10, themammalian cells are human cells.

Embodiment 12

In some further embodiments of any one of embodiments 4-11, the affinitymodified IgSF domain is capable of specifically binding to the twomammalian cells forming the IS.

Embodiment 13

In some further embodiments of any one of embodiments 1-12, thewild-type IgSF domain is from an IgSF family member of a family selectedfrom Signal-Regulatory Protein (SIRP) Family, Triggering ReceptorExpressed On Myeloid Cells Like (TREML) Family, CarcinoembryonicAntigen-related Cell Adhesion Molecule (CEACAM) Family, Sialic AcidBinding Ig-Like Lectin (SIGLEC) Family, Butyrophilin Family, B7 family,CD28 family, V-set and Immunoglobulin Domain Containing (VSIG) family,V-set transmembrane Domain (VSTM) family, Major HistocompatibilityComplex (MHC) family, Signaling lymphocytic activation molecule (SLAM)family, Leukocyte immunoglobulin-like receptor (LIR), Nectin (Nec)family, Nectin-like (NECL) family, Poliovirus receptor related (PVR)family, Natural cytotoxicity triggering receptor (NCR) family, T cellimmunoglobulin and mucin (TIM) family or Killer-cell immunoglobulin-likereceptors (KIR) family.

Embodiment 14

In some further embodiments of any one of embodiments 1-13, thewild-type IgSF domain is from an IgSF member selected from CD80, CD86,PD-L1, PD-L2, ICOS Ligand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA,CD4, CD8-alpha, CD8-beta, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2,CD226, CD2, CD160, CD200, CD200R or Nkp30.

Embodiment 15

In some further embodiments of any one of embodiments 1-14, thewild-type IgSF domain is a human IgSF member.

Embodiment 16

In some further embodiments of any one of embodiments 1-15, thewild-type IgSF domain is an IgV domain, an IgC1 domain, an IgC2 domainor a specific binding fragment thereof.

Embodiment 17

In some further embodiments of any one of embodiments 1-16, theaffinity-modified IgSF domain is an affinity modified IgV domain,affinity modified IgC1 domain or an affinity modified IgC2 domain or isa specific binding fragment thereof comprising the one or more aminoacid substitutions.

Embodiment 18

In some further embodiments of any one of embodiments 1-17, theimmunomodulatory protein comprises at least two non-immunoglobulinaffinity modified IgSF domains.

Embodiment 19

In some further embodiments of embodiment 18, the at least twonon-immunoglobulin affinity modified IgSF domains each comprise one ormore different amino acid substitutions in the same wild-type IgSFdomain.

Embodiment 20

In some further embodiments of embodiment 19, the at least twonon-immunoglobulin affinity modified IgSF domains each comprise one ormore amino acid substitutions in different wild-type IgSF domains.

Embodiment 21

In some further embodiments of embodiment 20, the different wild-typeIgSF domains are from different IgSF family members.

Embodiment 22

In some further embodiments of any one of embodiments 1-17, theimmunomodulatory protein comprises only one non-immunoglobulin affinitymodified IgSF domain.

Embodiment 23

In some further embodiments of any one of embodiments 1-22, theaffinity-modified IgSF comprises at least 85% sequence identity to awild-type IgSF domain or a specific binding fragment thereof containedin the sequence of amino acids set forth in any of SEQ ID NOS: 1-27.

Embodiment 24

In some further embodiments of embodiment 23, the immunomodulatoryprotein further comprises a second affinity-modified IgSF, wherein thesecond affinity-modified IgSF domain comprises at least 85% sequenceidentity to a wild-type IgSF domain or a specific binding fragmentthereof contained in the sequence of amino acids set forth in any of SEQID NOS: 1-27.

Embodiment 25

In some further embodiments of any one of embodiments 1-24, thewild-type IgSF domain is a member of the B7 family.

Embodiment 26

In some further embodiments of any one of embodiments 1-25, thewild-type IgSF domain is a domain of CD80, CD86 or ICOSLG.

Embodiment 27

In some further embodiments of any one of embodiments 1-26, thewild-type IgSF domain is a domain of CD80.

Embodiment 28

In some embodiments, there is provided an immunomodulatory protein,comprising at least one affinity modified CD80 immunoglobulinsuperfamily (IgSF) domain comprising one or more amino acidsubstitution(s) in a wild-type CD80 IgSF domain, wherein the at leastone affinity modified CD80 IgSF domain has increased binding to at leasttwo cognate binding partners compared to the wild-type CD80 IgSF domain.

Embodiment 29

In some further embodiments of embodiment 27 or embodiment 28, thecognate binding partners are CD28 and PD-L1.

Embodiment 30

In some further embodiments of any one of embodiments 27-29, thewild-type IgSF domain is an IgV domain and/or the affinity modified CD80domain is an affinity modified IgV domain.

Embodiment 31

In some further embodiments of any one of embodiments 27-30, theaffinity-modified domain comprises at least 85% sequence identity to awild-type CD80 domain or a specific binding fragment thereof containedin the sequence of amino acids set forth in SEQ ID NO:1.

Embodiment 32

In some further embodiments of any one of embodiments 1-31, the at leastone affinity modified IgSF domain comprises at least 1 and no more thantwenty amino acid substitutions in the wild-type IgSF domain.

Embodiment 33

In some further embodiments of any one of embodiments 1-32, the at leastone affinity modified IgSF domain comprises at least 1 and no more thanten amino acid substitutions in the wild-type IgSF domain.

Embodiment 34

In some further embodiments of any one of embodiments 1-33, the at leastone affinity modified IgSF domain comprises at least 1 and no more thanfive amino acid substitutions in the wild-type IgSF domain.

Embodiment 35

In some further embodiments of any one of embodiments 1-34, the affinitymodified IgSF domain has at least 120% of the binding affinity as itswild-type IgSF domain to each of the at least two cognate bindingpartners.

Embodiment 36

In some further embodiments of any one of embodiments 1-35, theimmunomodulatory protein further comprises a non-affinity modified IgSFdomain.

Embodiment 37

In some further embodiments of any one of embodiments 1-36, theimmunomodulatory protein is soluble.

Embodiment 38

In some further embodiments of any one of embodiments 1-37, theimmunomodulatory protein lacks a transmembrane domain or a cytoplasmicdomain.

Embodiment 39

In some further embodiments of any one of embodiments 1-38, theimmunomodulatory protein comprises only the extracellular domain (ECD)or a specific binding fragment thereof comprising the affinity modifiedIgSF domain.

Embodiment 40

In some further embodiments of any one of embodiments 1-39, theimmunomodulatory protein is glycosylated or pegylated.

Embodiment 41

In some further embodiments of any one of embodiments 1-40, theimmunomodulatory protein is linked to a multimerization domain.

Embodiment 42

In some further embodiments of any one of embodiments 1-41, theimmunomodulatory protein is linked to an Fc domain or a variant thereofwith reduced effector function.

Embodiment 43

In some further embodiments of embodiment 42, the Fc domain is an IgG1domain, an IgG2 domain or is a variant thereof with reduced effectorfunction.

Embodiment 44

In some further embodiments of any one of embodiments 39-41, the Fcdomain is mammalian, optionally human; or the variant Fc domaincomprises one or more amino acid modifications compared to an unmodifiedFc domain that is mammalian, optionally human.

Embodiment 45

In some further embodiments of any one of embodiments 42-44, the Fcdomain or variant thereof comprises the sequence of amino acids setforth in SEQ ID NO:226 or SEQ ID NO:227 or a sequence of amino acidsthat exhibits at least 85% sequence identity to SEQ ID NO:226 or SEQ IDNO:227.

Embodiment 46

In some further embodiments of any one of embodiments 38-41, theimmunomodulatory protein is linked indirectly via a linker.

Embodiment 47

In some further embodiments of any one of embodiments 41-46, theimmunomodulatory protein is a dimer.

Embodiment 48

In some further embodiments of any one of embodiments 1-47, theimmunomodulatory protein is attached to a liposomal membrane.

Embodiment 49

In some embodiments, there is provided an immunomodulatory protein,comprising at least two non-immunoglobulin immunoglobulin superfamily(IgSF) domains, wherein: at least one of the non-immunoglobulin modifiedIgSF domain is affinity-modified to exhibit altered binding to itscognate binding partner; and the at least two non-immunoglobulinmodified IgSF domain each independently specifically bind to at leastone different cognate binding partner.

Embodiment 50

In some further embodiments of embodiment 49, the each of the at leasttwo non-immunoglobulin IgSF domains are affinity-modified IgSF domains,wherein the first non-immunoglobulin modified IgSF domain comprises oneor more amino acid substitutions in a first wild-type-type IgSF domainand the second non-immunoglobulin modified IgSF domain comprises one ormore amino acid substitutions in a second wild-type IgSF domain.

Embodiment 51

In some further embodiments of embodiment 50, the firstnon-immunoglobulin modified IgSF domain exhibits altered binding to atleast one of its cognate binding partner(s) compared to the firstwild-type IgSF domain; and the second non-immunoglobulin modified IgSFdomain exhibits altered binding to at least one of its cognate bindingpartner(s) compared to the second wild-type IgSF domain.

Embodiment 52

In some further embodiments of any one of embodiments 49-51, thedifferent cognate binding partners are cell surface molecular speciesexpressed on the surface of a mammalian cell.

Embodiment 53

In some further embodiments of embodiment 52, the different cell surfacemolecular species are expressed in cis configuration or transconfiguration.

Embodiment 54

In some further embodiments of embodiment 52 or embodiment 53, themammalian cell is one of two mammalian cells forming an immunologicalsynapse (IS) and the different cell surface molecular species isexpressed on at least one of the two mammalian cells forming the IS.

Embodiment 55

In some further embodiments of any one of embodiments 52-54, at leastone of the mammalian cells is a lymphocyte.

Embodiment 56

In some further embodiments of embodiment 55, the lymphocyte is an NKcell or a T cell.

Embodiment 57

In some further embodiments of embodiment 55 or embodiment 56, bindingof the immunomodulatory protein to the cell modulates the immunologicalactivity of the lymphocyte.

Embodiment 58

In some further embodiments of embodiment 57, the immunomodulatoryprotein is capable of effecting increased immunological activitycompared to the wild-type protein comprising the wild-type IgSF domain.

Embodiment 59

In some further embodiments of embodiment 57, the immunomodulatoryprotein is capable of effecting decreased immunological activitycompared to the wild-type protein comprising the wild-type IgSF domain.

Embodiment 60

In some further embodiments of any one of embodiments 52-59, at leastone of the mammalian cells is a tumor cell.

Embodiment 61

In some further embodiments of any one of embodiments 52-60, themammalian cells are human cells.

Embodiment 62

In some further embodiments of any one of embodiments 54-61, theimmunomodulatory protein is capable of specifically binding to the twomammalian cells forming the IS.

Embodiment 63

In some further embodiments of any one of embodiments 49-62, the firstand second modified IgSF domains each comprise one or more amino acidsubstitutions in different wild-type IgSF domains.

Embodiment 64

In some further embodiments of embodiment 63, the different wild-typeIgSF domains are from different IgSF family members.

Embodiment 65

In some further embodiments of any one of embodiments 49-64, the firstand second modified IgSF domains are non-wild-type combinations.

Embodiment 66

In some further embodiments of any one of embodiments 49-65, the firstwild-type IgSF domain and second wild-type IgSF domain each individuallyis from an IgSF family member of a family selected fromSignal-Regulatory Protein (SIRP) Family, Triggering Receptor ExpressedOn Myeloid Cells Like (TREML) Family, Carcinoembryonic Antigen-relatedCell Adhesion Molecule (CEACAM) Family, Sialic Acid Binding Ig-LikeLectin (SIGLEC) Family, Butyrophilin Family, B7 family, CD28 family,V-set and Immunoglobulin Domain Containing (VSIG) family, V-settransmembrane Domain (VSTM) family, Major Histocompatibility Complex(MHC) family, Signaling lymphocytic activation molecule (SLAM) family,Leukocyte immunoglobulin-like receptor (LIR), Nectin (Nec) family,Nectin-like (NECL) family, Poliovirus receptor related (PVR) family,Natural cytotoxicity triggering receptor (NCR) family, T cellimmunoglobulin and mucin (TIM) family or Killer-cell immunoglobulin-likereceptors (KIR) family.

Embodiment 67

In some further embodiments of any one of embodiments 49-66, the firstwild-type IgSF domain and second wild-type IgSF domain each individuallyis from an IgSF member selected from CD80, CD86, PD-L1, PD-L2, ICOSLigand, B7-H3, B7-H4, CD28, CTLA4, PD-1, ICOS, BTLA, CD4, CD8-alpha,CD8-beta, LAG3, TIM-3, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160,CD200, CD200R or Nkp30.

Embodiment 68

In some further embodiments of any one of embodiments 49-67, the firstmodified IgSF domain and the second modified IgSF domain eachindividually comprises at least 85% sequence identity to a wild-typeIgSF domain or a specific binding fragment thereof contained in thesequence of amino acids set forth in any of SEQ ID NOS: 1-27.

Embodiment 69

In some further embodiments of any one of embodiments 49-68, the firstand second wild-type IgSF domain each individually is a member of the B7family.

Embodiment 70

In some further embodiments of embodiment 69, the first and secondwild-type IgSF domain each individually is from CD80, CD86 or ICOSLG.

Embodiment 71

In some further embodiments of any one of embodiments 49-68, the firstor second wild-type IgSF domain is from a member of the B7 family andthe other of the first or second wild-type IgSF domain is from anotherIgSF family.

Embodiment 72

In some further embodiments of any one of embodiments 49-68 and 71, thefirst and second wild-type IgSF domain is from ICOSLG and NKp30.

Embodiment 73

In some further embodiments of any one of embodiments 49-68 and 71, thefirst and second wild-type IgSF domain is from CD80 and NKp30.

Embodiment 74

In some further embodiments of any one of embodiments 49-73, the firstand second wild-type IgSF domain each individually is a human IgSFmember.

Embodiment 75

In some further embodiments of any one of embodiments 49-74, the firstand second wild-type IgSF domain each individually is an IgV domain, andIgC1 domain, an IgC2 domain or a specific binding thereof.

Embodiment 76

In some further embodiments of any one of embodiments 49-75, the firstnon-immunoglobulin modified domain and the second non-immunoglobulinmodified domain each individually is a modified IgV domain, modifiedIgC1 domain or a modified IgC2 domain or is a specific binding fragmentthereof comprising the one or more amino acid substitutions.

Embodiment 77

In some further embodiments of any one of embodiments 49-76, at leastone of the first non-immunoglobulin modified domain or the secondnon-immunoglobulin modified domain is a modified IgV domain.

Embodiment 78

In some further embodiments of any one of embodiments 49-77, the firstnon-immunoglobulin modified IgSF domain and the secondnon-immunoglobulin modified IgSF domain each individually comprise 1 andno more than twenty amino acid substitutions.

Embodiment 79

In some further embodiments of any one of embodiments 49-78, the firstnon-immunoglobulin modified IgSF domain and the secondnon-immunoglobulin modified IgSF domain each individually comprise 1 andno more than ten amino acid substitutions.

Embodiment 80

In some further embodiments of any one of embodiments 49-79, the firstnon-immunoglobulin modified IgSF domain and the secondnon-immunoglobulin modified IgSF domain each individually comprise 1 andno more than five amino acid substitutions.

Embodiment 81

In some further embodiments of any one of embodiments 49-80, at leastone of the first or second non-immunoglobulin modified IgSF domain hasbetween 10% and 90% of the binding affinity of the wild-type IgSF domainto at least one of its cognate binding partner.

Embodiment 82

In some further embodiments of any one of embodiments 49-81, at leastone of the first of second non-immunoglobulin modified IgSF domain hasat least 120% of the binding affinity of the wild-type IgSF domain to atleast one of its cognate binding partner.

Embodiment 83

In some further embodiments of any one of embodiments 49-80 and 82, thefirst and second non-immunoglobulin modified IgSF domain eachindividually has at least 120% of the binding affinity of the wild-typeIgSF domain to at least one of its cognate binding partner.

Embodiment 84

In some further embodiments of any one of embodiments 49-83, theimmunomodulatory protein is soluble.

Embodiment 85

In some further embodiments of any one of embodiments 49-84, theimmunomodulatory protein is glycosylated or pegylated.

Embodiment 86

In some further embodiments of any one of embodiments 49-85, theimmunomodulatory protein is linked to a multimerization domain.

Embodiment 87

In some further embodiments of any one of embodiments 49-86, theimmunomodulatory protein is linked to an Fc domain or a variant thereofwith reduced effector function.

Embodiment 88

In some further embodiments of embodiment 87, the Fc domain is an IgG1domain, an IgG2 domain or is a variant thereof with reduced effectorfunction.

Embodiment 89

In some further embodiments of embodiment 87 or embodiment 88, the Fcdomain is mammalian, optionally human; or the variant Fc domaincomprises one or more amino acid modifications compared to an unmodifiedFc domain that is mammalian, optionally human.

Embodiment 90

In some further embodiments of any one of embodiments 87-89, the Fcdomain or variant thereof comprises the sequence of amino acids setforth in SEQ ID NO:226 or SEQ ID NO:227 or a sequence of amino acidsthat exhibits at least 85% sequence identity to SEQ ID NO:226 or SEQ IDNO:227.

Embodiment 91

In some further embodiments of any one of embodiments 86-90, the variantCD80 polypeptide is linked indirectly via a linker.

Embodiment 92

In some further embodiments of any one of embodiments 86-91, theimmunomodulatory protein is a dimer.

Embodiment 93

In some further embodiments of any one of embodiments 49-92, theimmunomodulatory protein further comprises one or more additionalnon-immunoglobulin IgSF domain that is the same of different from thefirst or second non-immunoglobulin modified IgSF domain.

Embodiment 94

In some further embodiments of embodiment 93, the one or more additionalnon-immunoglobulin IgSF domain is an affinity modified IgSF domain.

Embodiment 95

In some further embodiments of any one of embodiment 49-94, theimmunomodulatory protein is attached to a liposomal membrane.

Embodiment 96

In some embodiments, there is provided a nucleic acid molecule, encodingthe immunomodulatory polypeptide of any of embodiments 1-95.

Embodiment 97

In some further embodiments of embodiment 96, the nucleic acid issynthetic nucleic acid.

Embodiment 98

In some further embodiments of embodiment 96 or embodiment 97, thenucleic acid is cDNA.

Embodiment 99

In some embodiments, there is provided a vector, comprising the nucleicacid molecule of any of embodiments 96-98.

Embodiment 100

In some further embodiments of embodiment 99, the vector is anexpression vector.

Embodiment 101

In some embodiments, there is provided a cell, comprising the vector ofembodiment 99 or embodiment 100.

Embodiment 102

In some further embodiments of embodiment 101, the cell is a eukaryoticcell or prokaryotic cell.

Embodiment 103

In some embodiments, there is provided a method of producing animmunomodulatory protein, comprising introducing the nucleic acidmolecule of any of embodiments 96-98 or vector of embodiment 99 orembodiment 100 into a host cell under conditions to express the proteinin the cell.

Embodiment 104

In some further embodiments of embodiment 103, the method furthercomprises isolating or purifying the immunomodulatory protein from thecell.

Embodiment 105

In some embodiments, there is provided a pharmaceutical composition,comprising the immunomodulatory protein of any of embodiments 1-95.

Embodiment 106

In some further embodiments of embodiment 105, the pharmaceuticalcomposition comprises a pharmaceutically acceptable excipient.

Embodiment 107

In some further embodiments of embodiment 105 or embodiment 106, thepharmaceutical composition is sterile.

Embodiment 108

In some embodiments, there is provided an article of manufacturecomprising the pharmaceutical composition of any of embodiments 105-107in a vial.

Embodiment 109

In some further embodiments of embodiment 108, the vial is sealed.

Embodiment 110

In some embodiments, there is provided a kit comprising thepharmaceutical composition of any of embodiments 105-107, andinstructions for use.

Embodiment 111

In some embodiments, there is provided a kit comprising the article ofmanufacture according to embodiment 108 or embodiment 109, andinstructions for use.

Embodiment 112

In some embodiments, there is provided a method of modulating an immuneresponse in a subject, comprising administering therapeuticallyeffective amount of the immunomodulatory protein of any of embodiments1-95 to the subject.

Embodiment 113

In some further embodiments of embodiment 112, modulating the immuneresponse treats a disease or condition in the subject.

Embodiment 114

In some further embodiments of embodiment 112 or embodiment 113, theimmune response is increased.

Embodiment 115

In some further embodiments of embodiment 114, the disease or conditionis a tumor or cancer.

Embodiment 116

In some further embodiments of embodiment 114 or embodiment 115, thedisease or condition is selected from melanoma, lung cancer, bladdercancer or a hematological malignancy.

Embodiment 117

In some further embodiments of embodiment 112 or embodiment 113, theimmune response is decreased.

Embodiment 118

In some further embodiments of embodiment 117, the disease or conditionis an inflammatory disease or condition.

Embodiment 119

In some further embodiments of embodiment 117 or embodiment 118, thedisease or condition is selected from Crohn's disease, ulcerativecolitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.

Embodiment 120

In some embodiments, there is provided a method of identifying anaffinity modified immunomodulatory protein, comprising: a) contacting amodified protein comprising at least one non-immunoglobulin modifiedimmunoglobulin superfamily (IgSF) domain or specific binding fragmentthereof with at least two cognate binding partners under conditionscapable of effecting binding of the protein with the at least twocognate binding partners, wherein the at least one modified IgSF domaincomprises one or more amino acid substitutions in a wild-type IgSFdomain; b) identifying a modified protein comprising the modified IgSFdomain that has increased binding to at least one of the two cognatebinding partners compared to a protein comprising the wild-type IgSFdomain; and c) selecting a modified protein comprising the modified IgSFdomain that binds non-competitively to the at least two cognate bindingpartners, thereby identifying the affinity modified immunomodulatoryprotein.

Embodiment 121

In some further embodiments of embodiment 120, step b) comprisesidentifying a modified protein comprising a modified IgSF domain thathas increased binding to each of the at least two cognate bindingpartners compared to a protein comprising the wild-type domain.

Embodiment 122

In some further embodiments of embodiment 120 or embodiment 121, priorto step a), introducing one or more amino acid substitutions into thewild-type IgSF domain, thereby generating a modified protein comprisingthe modified IgSF domain.

Embodiment 123

In some further embodiments of any one of embodiments 120-122, themodified protein comprises at least two modified IgSF domains orspecific binding fragments thereof, wherein the first IgSF domaincomprises one or more amino acid substitutions in a first wild-type-typeIgSF domain and the second non-immunoglobulin affinity modified IgSFdomain comprises one or more amino acid substitutions in a secondwild-type IgSF domain.

Embodiment 124

In some further embodiments of embodiment 123, the first and secondnon-immunoglobulin affinity modified IgSF domain each specifically bindto at least one different cognate binding partner.

Embodiment 125

In some embodiments, there is provided an immunomodulatory proteincomprising at least one non-immunoglobulin affinity-modifiedimmunoglobulin superfamily (IgSF) domain, wherein the affinity-modifiedIgSF domain specifically binds non-competitively to at least twocell-surface molecular species, wherein each of the molecular species isexpressed on at least one of the two mammalian cells forming animmunological synapse (IS), wherein one of the mammalian cells is alymphocyte and wherein binding of the affinity-modified IgSF domainmodulates immunological activity of the lymphocyte.

Embodiment 126

In some further embodiments of embodiment 125, the affinity modifiedIgSF domain specifically binds to the two mammalian cells forming theIS.

Embodiment 127

In some further embodiments of embodiment 125 or embodiment 126, theimmunomodulatory protein comprises at least two non-immunoglobulinaffinity modified IgSF domains and the immunomodulatory proteinspecifically binds to the two mammalian cells forming the IS.

Embodiment 128

In some further embodiments of any one of embodiments 125-127, the IgSFcell surface molecular species are human IgSF members.

Embodiment 129

In some further embodiments of any one of embodiments 125-128, theaffinity modified IgSF domain comprises at least one affinity modifiedhuman CD80 domain.

Embodiment 130

In some further embodiments of any one of embodiments 125-129, theimmunomodulatory protein comprises an affinity modified mammalian IgSFmember.

Embodiment 131

In some further embodiments of any one of embodiments 125-130, theaffinity modified mammalian IgSF member is at least one of: CD80, PVR,ICOSLG, or HAVCR2.

Embodiment 132

In some further embodiments of any one of embodiments 125-131,immunological activity is enhanced.

Embodiment 133

In some further embodiments of any one of embodiments 125-132,immunological activity is suppressed.

Embodiment 134

In some further embodiments of any one of embodiments 125-133, one ofthe two mammalian cells is a tumor cell.

Embodiment 135

In some further embodiments of any one of embodiments 125-134, thelymphocyte is an NK cell or a T-cell.

Embodiment 136

In some further embodiments of any one of embodiments 125-135, themammalian cells are a mouse, rat, cynomologus monkey, or human cells.

Embodiment 137

In some further embodiments of any one of embodiments 125-136, theaffinity modified IgSF domain has between 10% and 90% of the bindingaffinity of the wild-type IgSF domain to at least one of the two cellsurface molecular species.

Embodiment 138

In some further embodiments of any one of embodiments 125-137, theaffinity modified IgSF domain specifically binds non-competitively toexactly one IgSF member.

Embodiment 139

In some further embodiments of any one of embodiments 125-138, theaffinity modified IgSF domain has at least 120% of the binding affinityas its wild-type IgSF domain to at least one of the two cell surfacemolecular species.

Embodiment 140

In some further embodiments of any one of embodiments 125-139, theaffinity modified IgSF domain is an affinity modified IgV, IgC1, or IgC2domain.

Embodiment 141

In some further embodiments of any one of embodiments 125-140, theaffinity modified IgSF domain differs by at least one and no more thanten amino acid substitutions from its wild-type IgSF domain.

Embodiment 142

In some further embodiments of any one of embodiments 125-141, theaffinity modified IgSF domain differs by at least one and no more thanfive amino acid substitutions from its wild-type IgSF domain.

Embodiment 143

In some further embodiments of any one of embodiments 125-142, theaffinity modified IgSF domain is a human CD80 IgSF domain.

Embodiment 144

In some further embodiments of any one of embodiments 125-143, theimmunomodulatory protein comprises at least two affinity-modified IgSFdomains, wherein the affinity modified IgSF domains are not the samespecies of IgSF domain.

Embodiment 145

In some further embodiments of any one of embodiments 125-144, theimmunomodulatory protein is covalently bonded, directly or indirectly,to an antibody fragment crystallizable (Fc).

Embodiment 146

In some further embodiments of any one of embodiments 125-145, theimmunomodulator protein is in a pharmaceutically acceptable carrier.

Embodiment 147

In some further embodiments of any one of embodiments 125-146, theimmunomodulatory protein is glycosylated or pegylated.

Embodiment 148

In some further embodiments of any one of embodiments 125-147, theimmunomodulatory protein is soluble.

Embodiment 149

In some further embodiments of any one of embodiments 125-148, theimmunomodulatory protein is attached to a liposomal membrane.

Embodiment 150

In some further embodiments of any one of embodiments 125-149, theimmunomodulatory protein is dimerized by intermolecular disulfide bonds.

Embodiment 151

In some further embodiments of any one of embodiments 125-150, the cellsurface molecular species are expressed in cis configuration or transconfiguration.

Embodiment 152

In some embodiments, there is provided an immunomodulatory proteincomprising at least two non-immunoglobulin affinity-modifiedimmunoglobulin superfamily (IgSF) domains, wherein the affinity-modifiedIgSF domains each specifically binds to a different cell surfacemolecular species, wherein each of the molecular species is expressed onat least one of the two mammalian cells forming an immunological synapse(IS), wherein one of the mammalian cells is a lymphocyte, and whereinbinding of the affinity-modified IgSF domain modulates immunologicalactivity of the lymphocyte.

Embodiment 153

In some further embodiments of any one of embodiments 125-152, at leastone of the affinity modified IgSF domains binds competitively.

Embodiment 154

In some further embodiments of any one of embodiments 125-153, theaffinity modified IgSF domains are not the same species of IgSF domain.

Embodiment 155

In some further embodiments of any one of embodiments 125-154, theaffinity modified IgSF domains are non-wild type combinations.

Embodiment 156

In some further embodiments of any one of embodiments 125-155, the cellsurface molecular species are human IgSF members.

Embodiment 157

In some further embodiments of any one of embodiments 125-156, the atleast two affinity modified IgSF domains are from at least one of: CD80,CD86, CD274, PDCD1LG2, ICOSLG, CD276, VTCN1, CD28, CTLA4, PDCD1, ICOS,BTLA, CD4, CD8A, CD8B, LAG3, HAVCR2, CEACAM1, TIGIT, PVR, PVRL2, CD226,CD2, CD160, CD200, or CD200R1.

Embodiment 158

In some further embodiments of any one of embodiments 125-157, theimmunomodulatory protein comprises at least two affinity modifiedmammalian IgSF members.

Embodiment 159

In some further embodiments of any one of embodiments 125-158, themammalian IgSF members are human IgSF members.

Embodiment 160

In some further embodiments of any one of embodiments 125-159, themammalian IgSF members are at least two of: CD80, CD86, CD274, PDCD1LG2,ICOSLG, CD276, VTCN1, CD28, CTLA4, PDCD1, ICOS, BTLA, CD4, CD8A, CD8B,LAG3, HAVCR2, CEACAM1, TIGIT, PVR, PVRL2, CD226, CD2, CD160, CD200, orCD200R1.

Embodiment 161

In some further embodiments of any one of embodiments 125-160,immunological activity is enhanced.

Embodiment 38

In some further embodiments of any one of embodiments 125-1,immunological activity is suppressed.

Embodiment 162

In some further embodiments of any one of embodiments 125-161, one ofthe two mammalian cells is a tumor cell.

Embodiment 163

In some further embodiments of any one of embodiments 125-162, thelymphocyte is an NK cell or a T-cell.

Embodiment 164

In some further embodiments of any one of embodiments 125-163, themammalian cells are a mouse, rat, cynomologus monkey, or human cells.

Embodiment 165

In some further embodiments of any one of embodiments 125-164, at leastone of the two affinity modified IgSF domains has between 10% and 90% ofthe binding affinity of the wild-type IgSF domain to at least one of thecell surface molecular species.

Embodiment 166

In some further embodiments of any one of embodiments 125-165, at leastone of the two affinity modified IgSF domains specifically binds toexactly one cell surface molecular species.

Embodiment 167

In some further embodiments of any one of embodiments 125-166, at leastone of the two affinity modified IgSF domains has at least 120% of thebinding affinity as its wild-type IgSF domain to at least one of the twocell surface molecular species.

Embodiment 168

In some further embodiments of any one of embodiments 125-167, theaffinity modified IgSF domains are at least one of an IgV, IgC1, or IgC2domain.

Embodiment 169

In some further embodiments of any one of embodiments 125-168, each ofthe at least two affinity modified IgSF domains differs by at least oneand no more than ten amino acid substitutions from its wild-type IgSFdomain.

Embodiment 170

In some further embodiments of any one of embodiments 125-169, each ofthe at least two affinity modified IgSF domains differs by at least oneand no more than five amino acid substitutions from its wild-type IgSFdomain.

Embodiment 171

In some further embodiments of any one of embodiments 125-170, theimmunomodulatory protein is covalently bonded, directly or indirectly,to an antibody fragment crystallizable (Fc).

Embodiment 172

In some further embodiments of any one of embodiments 125-171, theimmunomodulatory protein is in a pharmaceutically acceptable carrier.

Embodiment 173

In some further embodiments of any one of embodiments 125-172, theprotein is glycosylated or pegylated.

Embodiment 174

In some further embodiments of any one of embodiments 125-173, theprotein is soluble.

Embodiment 175

In some further embodiments of any one of embodiments 125-174, theprotein is bonded to a liposomal membrane.

Embodiment 176

In some further embodiments of any one of embodiments 125-175, theprotein is dimerized by intermolecular disulfide bonds.

Embodiment 177

In some further embodiments of any one of embodiments 125-176, the cellsurface molecular species are expressed in cis configuration or transconfiguration.

Embodiment 178

In some further embodiments of any one of embodiments 125-177, theimmunomodulatory protein has at least 85% sequence identity with anamino acid sequence selected from SEQ ID NOS: 1-26, a combination, or afragment thereof.

Embodiment 179

In some further embodiments of any one of embodiments 125-178, theimmunomodulatory protein has at least 90% sequence identity with anamino acid sequence selected from SEQ ID NOS: 1-26, a combination, or afragment thereof.

Embodiment 180

In some further embodiments of any one of embodiments 125-179, theimmunomodulatory protein has at least 95% sequence identity with anamino acid sequence selected from SEQ ID NOS: 1-26, a combination, or afragment thereof.

Embodiment 181

In some further embodiments of any one of embodiments 125-180, theimmunomodulatory protein has at least 99% sequence identity with anamino acid sequence selected from SEQ ID NOS: 1-26, a combination, or afragment thereof.

Embodiment 182

In some further embodiments of any one of embodiments 125-181, theimmunomodulatory protein further comprises a second immunomodulatoryprotein, wherein the second immunomodulatory protein has at least 85%sequence identity with an amino acid sequence selected from SEQ ID NOS:1-26 or a fragment thereof.

Embodiment 183

In some further embodiments of any one of embodiments 125-182, theimmunomodulatory protein further comprises a second immunomodulatoryprotein, wherein the second immunomodulatory protein has at least 90%sequence identity with an amino acid sequence selected from SEQ ID NOS:1-26 or a fragment thereof.

Embodiment 184

In some further embodiments of any one of embodiments 125-183, theimmunomodulatory protein further comprises a second immunomodulatoryprotein, wherein the second immunomodulatory protein has at least 95%sequence identity with an amino acid sequence selected from SEQ ID NOS:1-26 or a fragment thereof.

Embodiment 185

In some further embodiments of any one of embodiments 125-184, theimmunomodulatory protein further comprises a second immunomodulatoryprotein, wherein the second immunomodulatory protein has at least 99%sequence identity with an amino acid sequence selected from SEQ ID NOS:1-26 or a fragment thereof.

Embodiment 186

In some embodiments, there is provided a recombinant nucleic acidencoding any one of the immunomodulatory proteins of embodiments125-185.

Embodiment 187

In some embodiments, there is provided a recombinant expression vectorcomprising a nucleic acid of embodiment 186.

Embodiment 188

In some embodiments, there is provided a recombinant host cellcomprising the expression vector of embodiment 187.

Embodiment 189

In some embodiments, there is provided a method of making animmunomodulatory protein of any one of embodiments 125-185, comprisingculturing the recombinant host cell under immunomodulatory proteinexpressing conditions, expressing the immunomodulatory protein encodedby the recombinant expression vector therein, and purifying therecombinant immunomodulatory protein expressed thereby.

Embodiment 190

In some embodiments, there is provided a method of treating a mammalianpatient in need of an enhanced or suppressed immunological response byadministering a therapeutically effective amount of immunomodulatoryprotein of any one of embodiments 125-185.

Embodiment 191

In some further embodiments of embodiment 190, the enhancedimmunological response treats melanoma, lung cancer, bladder cancer, ora hematological malignancy in the patient.

Embodiment 192

In some further embodiments of embodiment 190, the suppressedimmunological response treats Crohn's disease, ulcerative colitis,multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis in thepatient.

VIII. Examples

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Examples 1-8 describe the design, creation, and screening of affinitymodified CD80 (B7-1), CD86 (B7-2), ICOSL, and NKp30 immunomodulatoryproteins, which are components of the immune synapse (IS) that have ademonstrated dual role in both immune activation and inhibition. Theseexamples demonstrate that affinity modification of IgSF domains yieldsproteins that can act to both increase and decrease immunologicalactivity. This work also describes the various combinations of thosedomains fused in pairs (i.e., stacked) to form a Type IIimmunomodulatory protein to achieve immunomodulatory activity.

Example 1 Generation of Mutant DNA Constructs of IgSF Domains

Example 1 describes the generation of mutant DNA constructs of humanCD80, CD86, ICOSL, and NKp30 IgSF domains for translation and expressionon the surface of yeast as yeast display libraries.

A. Degenerate Libraries

For libraries that target specific residues of target protein forcomplete or partial randomization with degenerate codons, the codingDNA's for the extracellular domains (ECD) of human CD80 (SEQ ID NO:28),ICOSL (SEQ ID NO:32), and NKp30 (SEQ ID NO:54) were ordered fromIntegrated DNA Technologies (Coralville, Iowa) as a set of overlappingoligonucleotides of up to 80 base pairs (bp) in length. To generate alibrary of diverse variants of each ECD, the oligonucleotides containeddesired degenerate codons at desired amino acid positions. Degeneratecodons were generated using an algorithm at the URL:rosettadesign.med.unc.edu/SwiftLib/.

In general, positions to mutate and degenerate codons were chosen asfollows: crystal structures (CD80, NKp30) or homology models (ICOSL) ofthe target-ligand pairs of interest were used to identify ligand contactresidues as well as residues that are at the protein interactioninterface. This analysis was performed using a structure vieweravailable at the URL:spdbv.vital-it.ch). For example, a crystalstructure for CD80 bound to CTLA4 is publicly available at theURL:www.rcsb.org/pdb/explore/explore.do?structureId=1I8L) and a targetedlibrary was designed based on the CD80::CTLA4 interface for selection ofimproved binders to CTLA4. However, there are no CD80 structuresavailable with ligands CD28 and PDL1, so the same library was also usedto select for binders of CD28 (binds the same region on CD80 as CTLA4)and PDL1 (not known if PDL1 binds the same site as CTLA4).

The next step in library design was the alignment of human, mouse, ratand monkey CD80, ICOSL or NKp30 sequences to identify conservedresidues. Based on this analysis, conserved target residues were mutatedwith degenerate codons that only specified conservative amino acidchanges plus the wild-type residue. Residues that were not conserved,were mutated more aggressively, but also including the wild-typeresidue. Degenerate codons that also encoded the wild-type residue weredeployed to avoid excessive mutagenesis of target protein. For the samereason, only up to 20 positions were targeted for mutagenesis at a time.These residues were a combination of contact residues and non-contactinterface residues.

The oligonucleotides were dissolved in sterile water, mixed in equimolarratios, heated to 95° C. for five minutes and slowly cooled to roomtemperature for annealing. ECD-specific oligonucleotide primers thatanneal to the start and end of the ECDs, respectively, were then used togenerate PCR product. ECD-specific oligonucleotides which overlap by40-50 bp with a modified version of pBYDS03 cloning vector (LifeTechnologies USA), beyond and including the BamH1 and Kpn1 cloningsites, were then used to amplify 100 ng of PCR product from the priorstep to generate a total of 5 μg of DNA. Both PCR's were by polymerasechain reaction (PCR) using OneTaq 2×PCR master mix (New England Biolabs,USA). The second PCR products were purified using a PCR purification kit(Qiagen, Germany) and resuspended in sterile deionized water.

To prepare for library insertion, a modified yeast display version ofvector pBYDS03 was digested with BamH1 and Kpn1 restriction enzymes (NewEngland Biolabs, USA) and the large vector fragment was gel-purified anddissolved in sterile, deionized water. Electroporation-ready DNA for thenext step was generated by mixing 12 μg of library DNA with 4 μg oflinearized vector in a total volume of 50 μl deionized and sterilewater. An alternative way to generate targeted libraries, was to carryout site-directed mutagenesis (Multisite kit, Agilent, USA) of targetECD's with oligonucleotides containing degenerate codons. This approachwas used to generate sublibraries that only target specific stretches oftarget protein for mutagenesis. In these cases, sublibraries were mixedbefore proceeding to the selection steps. In general, library sizes werein the range of 10E7 to 10E8 clones, except that sublibraries were onlyin the range of 10E4 to 10E5. Large libraries were generated for CD80,ICOSL, CD86 and NKp30. Sublibraries were generated for CD80, ICOSL andNKp30.

B. Random Libraries

Random libraries were also constructed to identify variants of the ECDof CD80 (SEQ ID NO:28), CD86 (SEQ ID NO: 29), ICOSL (SEQ ID NO:32) andNKp30 (SEQ ID NO:54. DNA encoding wild-type ECDs was cloned between theBamH1 and Kpn1 sites of modified yeast display vector pBYDS03 and thenreleased using the same restriction enzymes. The released DNA was thenmutagenized with the Genemorph II kit (Agilent, USA) so as to generatean average of three to five amino acid changes per library variant.Mutagenized DNA was then amplified by the two-step PCR and furtherprocessed as described above for targeted libraries.

Example 2 Introduction of DNA Libraries into Yeast

Example 2 describes the introduction of CD80, CD86, ICOSL, and NKp30 DNAlibraries into yeast.

To introduce degenerate and random library DNA into yeast,electroporation-competent cells of yeast strain BJ5464 (ATCC.org; ATCCnumber 208288) were prepared and electroporated on a Gene Pulser II(Biorad, USA) with the electroporation-ready DNA from the step aboveessentially as described (Colby, D. W. et al. 2004 Methods Enzymology388, 348-358). The only exception is that transformed cells were grownin non-inducing minimal selective SCD-Leu medium to accommodate the LEU2selective marker carried by modified plasmid pBYDS03.

Library size was determined by plating dilutions of freshly recoveredcells on SCD-Leu agar plates and then extrapolating library size fromthe number of single colonies from plating that generated at least 50colonies per plate. The remainder of the electroporated culture wasgrown to saturation and cells from this culture were subcultured intothe same medium once more to minimize the fraction of untransformedcells. To maintain library diversity, this subculturing step was carriedout using an inoculum that contained at least 10× more cells than thecalculated library size. Cells from the second saturated culture wereresuspended in fresh medium containing sterile 25% (weight/volume)glycerol to a density of 10E10/ml and frozen and stored at −80° C.(frozen library stock).

One liter of SCD-Leu media consists of 14.7 grams of sodium citrate,4.29 grams of citric acid monohydrate, 20 grams of dextrose, 6.7 gramsof Difco brand yeast nitrogen base, and 1.6 grams yeast syntheticdrop-out media supplement without leucine. Media was filtered sterilizedbefore use using a 0.2 μM vacuum filter device.

Library size was determined by plating dilutions of freshly recoveredcells on SCD-Leu agar plates and then extrapolating library size fromthe number of single colonies from a plating that generate at least 50colonies per plate.

To segregate plasmid from cells that contain two or more differentlibrary clones, a number of cells corresponding to 10 times the librarysize, were taken from the overnight SCD-Leu culture and subcultured1/100 into fresh SCD-Leu medium and grown overnight. Cells from thisovernight culture were resuspended in sterile 25% (weight/volume)glycerol to a density of 10E10/ml and frozen and stored at −80° C.(frozen library stock).

Example 3 Yeast Selection

Example 3 describes the selection of yeast expressing affinity modifiedvariants of CD80, CD86, ICOSL, and NKp30.

A number of cells equal to at least 10 times the library size werethawed from individual library stocks, suspended to 0.1×10E6 cells/ml innon-inducing SCD-Leu medium, and grown overnight. The next day, a numberof cells equal to 10 times the library size were centrifuged at 2000 RPMfor two minutes and resuspended to 0.5×10E6 cells/ml in inducingSCDG-Leu media. One liter of the SCDG-Leu induction media consists of5.4 grams Na₂HPO₄, 8.56 grams of NaH₂PO₄*H₂O, 20 grams galactose, 2.0grams dextrose, 6.7 grams Difco yeast nitrogen base, and 1.6 grams ofyeast synthetic drop out media supplement without leucine dissolved inwater and sterilized through a 0.22 μm membrane filter device. Theculture was grown for two days at 20° C. to induce expression of libraryproteins on the yeast cell surface.

Cells were processed with magnetic beads to reduce non-binders andenrich for all CD80, CD86, ICOSL or NKp30 variants with the ability tobind their exogenous recombinant counter-structure proteins. Forexample, yeast displayed targeted or random CD80 libraries were selectedagainst each of CD28, CTL-4, PD-L1, ICOS, and B7-H6 separately. This wasthen followed by two to three rounds of flow cytometry sorting usingexogenous counter-structure protein staining to enrich the fraction ofyeast cells that displays improved binders. Magnetic bead enrichment andselections by flow cytometry are essentially as described in Keith D.Miller,1 Noah B. Pefaur,2 and Cheryl L. Baird1 Current Protocols inCytometry 4.7.1-4.7.30, July 2008.

With CD80, CD86, ICOSL, and NKp30 libraries, target ligand proteins weresourced from R&D Systems (USA) as follows: human rCD28.Fc (i.e.,recombinant CD28-Fc fusion protein), rPDL1.Fc, rCTLA4.Fc, rICOS.Fc, andrB7H6.Fc. Magnetic streptavidin beads were obtained from New EnglandBiolabs, USA. For biotinylation of counter-structure protein,biotinylation kit cat #21955, Life Technologies, USA, was used. Fortwo-color, flow cytometric sorting, a Becton Dickinson FACS Aria IIsorter was used. CD80, CD86, ICOSL, or NKp30 display levels weremonitored with an anti-hemagglutinin antibody labeled with Alexafluor488 (Life Technologies, USA). Ligand binding Fc fusion proteinsrCD28.Fc, rCTLA4.Fc, rPDL1.Fc, rICOS.Fc, or rB7-H6.Fc were detected withPE conjugated human Ig specific goat Fab (Jackson ImmunoResearch, USA).Doublet yeast were gated out using forward scatter (FSC)/side scatter(SSC) parameters, and sort gates were based upon higher ligand bindingdetected in FL4 that possessed more limited tag expression binding inFL1.

Yeast outputs from the flow cytometric sorts were assayed for higherspecific binding affinity. Sort output yeast were expanded andre-induced to express the particular IgSF affinity modified domainvariants they encode. This population then can be compared to theparental, wild-type yeast strain, or any other selected outputs, such asthe bead output yeast population, by flow cytometry.

For ICOSL, the second sort outputs (F2) were compared to parental ICOSLyeast for binding of each rICOS.Fc, rCD28.Fc, and rCTLA4.Fc by doublestaining each population with anti-HA (hemagglutinin) tag expression andthe anti-human Fc secondary to detect ligand binding.

In the case of ICOSL yeast variants selected for binding to ICOS, the F2sort outputs gave Mean Fluorescence Intensity (MFI) values of 997, whenstained with 5.6 nM rICOS.Fc, whereas the parental ICOSL strain MFI wasmeasured at 397 when stained with the same concentration of rICOS.Fc.This represents a roughly three-fold improvement of the average bindingin this F2 selected pool of clones, and it is predicted that individualclones from that pool will have much better improved MFI/affinity whenindividually tested.

In the case of ICOSL yeast variants selected for binding to CD28, the F2sort outputs gave MFI values of 640 when stained with 100 nM rCD28.Fc,whereas the parental ICOSL strain MFI was measured at 29 when stainedwith the same concentration of rCD28.Fc (22-fold improvement). In thecase of ICOSL yeast variants selected for binding to CTLA4, the F2 sortoutputs gave MFI values of 949 when stained with 100 nM rCTLA4.Fc,whereas the parental ICOSL strain MFI was measured at 29 when stainedwith the same concentration of rCTLA4.Fc (32-fold improvement).

In the case of NKp30 yeast variants selected for binding to B7-H6, theF2 sort outputs gave MFI values of 533 when stained with 16.6 nMrB7H6.Fc, whereas the parental NKp30 strain MFI was measured at 90 whenstained with the same concentration of rB7H6.Fc (6-fold improvement).

Importantly, the MFIs of all F2 outputs described above when measuredwith the anti-HA tag antibody on FL1 did not increase and sometimes wentdown compared to wild-type strains, indicating that increased bindingwas not a function of increased expression of the selected variants onthe surface of yeast, and validated gating strategies of only selectingmid to low expressors with high ligand binding.

Example 4 Reformatting Selection Outputs as Fc-Fusions and in VariousImmunomodulatory Protein Types

Example 4 describes reformatting of selection outputs asimmunomodulatory proteins containing an affinity modified (variant)extracellular domain (ECD) of CD80 or ICOSL, fused to an Fc molecule(variant ECD-Fc fusion molecules).

Output cells from final flow cytometric CD80 and ICOSL sorts were grownto terminal density in SCD-Leu medium. Plasmid DNA's from each outputwere isolated using a yeast plasmid DNA isolation kit (Zymoresearch,USA). For Fc fusions, PCR primers with added restriction sites suitablefor cloning into the Fc fusion vector of choice were used tobatch-amplify from the plasmid DNA preps the coding DNA's for the mutanttarget ECD's. After restriction digestion, the PCR products were ligatedinto an appropriate Fc fusion vector followed by chemical transformationinto strain XL1 Blue E. Coli (Agilent, USA) or NEB5alpha (New EnglandBiolabs) as directed by supplier. Exemplary of an Fc fusion vector ispFUSE-hIgG1-Fc2 (Invivogen, USA).

Dilutions of transformation reactions were plated on LB-agar containing100 μg/ml carbenicillin (Teknova, USA) to generate single colonies. Upto 96 colonies from each transformation were then grown in 96 wellplates to saturation overnight at 37 C in LB-broth (Teknova cat #L8112)and a small aliquot from each well was submitted for DNA sequencing ofthe ECD insert in order to identify the mutation(s) in all clones.Sample preparation for DNA sequencing was carried out using protocolsprovided by the service provider (Genewiz; South Plainfield, N.J.).After removal of sample for DNA sequencing, glycerol was then added tothe remaining cultures for a final glycerol content of 25% and plateswere stored at −20° C. for future use as master plates (see below).Alternatively, samples for DNA sequencing were generated by replicaplating from grown liquid cultures to solid agar plates using adisposable 96 well replicator (VWR, USA). These plates were incubatedovernight to generate growth patches and the plates were submitted toGenewiz as specified by Genewiz.

After identification of clones of interest from analysis ofGenewiz-generated DNA sequencing data, clones of interest were recoveredfrom master plates and individually grown to density in 5 ml liquidLB-broth containing 100 μg/ml carbenicillin (Teknova, USA) and 2 ml ofeach culture were then used for preparation of approximately 10 μg ofminiprep plasmid DNA of each clone using a standard kit such as thePureyield kit (Promega). Identification of clones of interest generallyinvolved the following steps. First, DNA sequence data files weredownloaded from the Genewiz website. All sequences were then manuallycurated so that they start at the beginning of the ECD coding region.The curated sequences were then batch-translated using a suitableprogram available at the URL: www.ebi.ac.uk/Tools/st/emboss_transeq/.The translated sequences were then aligned using a suitable programavailable at the URL:multalin.toulouse.inra.fr/multalin/multalin.html.

Clones of interest were then identified using the following criteria:1.) identical clone occurs at least two times in the alignment and 2.) amutation occurs at least two times in the alignment and preferably indistinct clones. Clones that meet at least one of these criteria wereclones that have been enriched by our sorting process due to improvedbinding.

To generate immunomodulatory proteins containing an ECD of CD80 or ICOSLwith at least one affinity-modified domain, the encoding nucleic acidmolecule was generated to encode a protein designed as follows: signalpeptide followed by variant (mutant) ECD followed by a linker of threealanines (AAA) followed by a human IgG1 Fc containing the mutation N82Gwith reference to wild-type human IgG1 Fc set forth in SEQ ID NO: 226.Since the construct does not include any antibody light chains that canform a covalent bond with a cysteine, the human IgG1 Fc also containsreplacement of the cysteine residues to a serine residue at position 5(C5S) compared to the wild-type or unmodified Fc set forth in SEQ ID NO:226.

In addition, Example 8 below describes further immunomodulatory proteinsthat were generated as stack constructs containing at least twodifferent affinity modified domains from identified variant CD80, CD86,ICOSL, and NKp30 molecules linked together and fused to an Fc.

Example 5 Expression and Purification of Fc-Fusions

Example 5 describes the high throughput expression and purification ofFc-fusion proteins containing variant ECD CD80, CD86, ICOSL, and Nkp30.

Recombinant variant Fc fusion proteins were produced with Expi293expression system (Invitrogen, USA). 4 μg of each plasmid DNA from theprevious step was added to 200 μl Opti-MEM (Invitrogen, USA) at the sametime as 10.8 μl ExpiFectamine was separately added to another 200 μlOpti-MEM. After 5 minutes, the 200 μl of plasmid DNA was mixed with the200 μl of ExpiFectamine and was further incubated for an additional 20minutes before adding this mixture to cells. Ten million Expi293 cellswere dispensed into separate wells of a sterile 10 ml, conical bottom,deep 24 well growth plate (Thomson Instrument Company, USA) in a volume3.4 ml Expi293 media (Invitrogen, USA). Plates were shaken for 5 days at120 RPM in a mammalian cell culture incubator set to 95% humidity and 8%CO₂. Following a 5 day incubation, cells were pelleted and culturesupernatants were removed.

Protein was purified from supernatants using a high throughput 96 wellProtein A purification kit using the manufacturer's protocol (Catalognumber 45202, Life Technologies, USA). Resulting elution fractions werebuffer exchanged into PBS using Zeba 96 well spin desalting plate(Catalog number 89807, Life Technologies, USA) using the manufacturer'sprotocol. Purified protein was quantitated using 280 nm absorbancemeasured by Nanodrop instrument (Thermo Fisher Scientific, USA), andprotein purity was assessed by loading 5 μg of protein on NUPAGEpre-cast, polyacrylamide gels (Life Technologies, USA) under denaturingand reducing conditions and subsequent gel electrophoresis. Proteinswere visualized in gel using standard Coomassie staining.

Example 6 Assessment of Binding and Activity of Affinity-Matured IgSFDomain-Containing Molecules

A. Binding to Cell-Expressed Counter Structures

This Example describes Fc-fusion binding studies to show specificity andaffinity of CD80 and ICOSL domain variant immunomodulatory proteins forcognate binding partners.

To produce cells expressing cognate binding partners, full-lengthmammalian surface expression constructs for each of human CD28, CTLA4,PD-L1, ICOS, and B7-H6 were designed in pcDNA3.1 expression vector (LifeTechnologies) and sourced from Genscript, USA. Binding studies werecarried out using the Expi293F transient transfection system (LifeTechnologies, USA) described above. The number of cells needed for theexperiment was determined, and the appropriate 30 ml scale oftransfection was performed using the manufacturer's suggested protocol.For each CD28, CTLA-4, PD-L1, ICOS, B7-H6 or mock 30 ml transfection, 75million Expi293F cells were incubated with 30 μg expression constructDNA and 1.5 ml diluted ExpiFectamine 293 reagent for 48 hours, at whichpoint cells were harvested for staining.

For staining by flow cytometry, 200,000 cells of appropriate transienttransfection or negative control were plated in 96 well round bottomplates. Cells were spun down and resuspended in staining buffer (PBS(phosphate buffered saline), 1% BSA (bovine serum albumin), and 0.1%sodium azide) for 20 minutes to block non-specific binding. Afterwards,cells were centrifuged again and resuspended in staining buffercontaining 100 nM to 1 nM variant immunomodulatory protein, depending onthe experiment of each candidate CD80 variant Fc, ICOSL variant Fc, orstacked IgSF variant Fc fusion protein in 50 μl. Primary staining wasperformed on ice for 45 minutes, before washing cells in staining buffertwice. PE-conjugated anti-human Fc (Jackson ImmunoResearch, USA) wasdiluted 1:150 in 50 μl staining buffer and added to cells and incubatedanother 30 minutes on ice. Secondary antibody was washed out twice,cells were fixed in 4% formaldehyde/PBS, and samples were analyzed onFACScan flow cytometer (Becton Dickinson, USA).

Mean Fluorescence Intensity (MFI) was calculated for each transfectantand negative parental line with Cell Quest Pro software (BectonDickinson, USA).

B. Bioactivity Characterization

This Example further describes Fc-fusion variant protein bioactivitycharacterization in human primary T cell in vitro assays.

1. Mixed Lymphocyte Reaction (MLR)

Soluble rICOSL.Fc or rCD80.Fc bioactivity was tested in a human MixedLymphocyte Reaction (MLR). Human primary dendritic cells (DC) weregenerated by culturing monocytes isolated from PBMC (BenTech Bio, USA)in vitro for 7 days with 500 U/ml rIL-4 (R&D Systems, USA) and 250 U/mlrGM-CSF (R&D Systems, USA) in Ex-Vivo 15 media (Lonza, Switzerland).10,000 matured DC and 100,000 purified allogeneic CD4+ T cells (BenTechBio, USA) were co-cultured with ICOSL or CD80 variant Fc fusion proteinsand controls in 96 well round bottom plates in 200 μl final volume ofEx-Vivo 15 media. On day 5, IFN-gamma secretion in culture supernatantswas analyzed using the Human IFN-gamma Duoset ELISA kit (R&D Systems,USA). Optical density was measured by VMax ELISA Microplate Reader(Molecular Devices, USA) and quantitated against titrated rIFN-gammastandard included in the IFN-gamma Duo-set kit (R&D Systems, USA).

2. Anti-CD3 Coimmobilization Assay

Costimulatory bioactivity of ICOSL and CD80 Fc fusion variants wasdetermined in anti-CD3 coimmobilization assays. 1 nM or 4 nM mouseanti-human CD3 (OKT3, Biolegends, USA) was diluted in PBS with 1 nM to80 nM rICOSL.Fc or rCD80.Fc variant proteins. This mixture was added totissue culture treated flat bottom 96 well plates (Corning, USA)overnight to facilitate adherence of the stimulatory proteins to thewells of the plate. The next day, unbound protein was washed off theplates and 100,000 purified human pan T cells (BenTech Bio, US) or humanT cell clone BC3 (Astarte Biologics, USA) were added to each well in afinal volume of 200 μl of Ex-Vivo 15 media (Lonza, Switzerland). Cellswere cultured 3 days before harvesting culture supernatants andmeasuring human IFN-gamma levels with Duoset ELISA kit (R&D Systems,USA) as mentioned above.

C. Results

Results for the binding and activity studies for exemplary testedvariants are shown in Tables 6-8. In particular, Table 6 indicatesexemplary IgSF domain amino acid substitutions (replacements) in the ECDof CD80 selected in the screen for affinity-maturation against therespective cognate structure CD28. Table 7 indicates exemplary IgSFdomain amino acid substitutions (replacements) in the ECD of CD80selected in the screen for affinity-maturation against the respectivecognate structure PD-L1. Table 8 indicates exemplary IgSF domain aminoacid substitutions (replacements) in the ECD of ICOSL selected in thescreen for affinity-maturation against the respective cognate structuresICOS and CD28. For each Table, the exemplary amino acid substitutionsare designated by amino acid position number corresponding to therespective reference unmodified ECD sequence as follows. For example,the reference unmodified ECD sequence in Tables 6 and 7 is theunmodified CD80 ECD sequence set forth in SEQ ID NO:28 and the referenceunmodified ECD sequence in Table 8 is the unmodified ICOSL ECD sequence(SEQ ID NO:32). The amino acid position is indicated in the middle, withthe corresponding unmodified (e.g. wild-type) amino acid listed beforethe number and the identified variant amino acid substitution listedafter the number. Column 2 sets forth the SEQ ID NO identifier for thevariant ECD for each variant ECD-Fc fusion molecule.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each variant Fc-fusion molecule tocells engineered to express the cognate counter structure ligand and theratio of the MFI compared to the binding of the corresponding unmodifiedECD-Fc fusion molecule not containing the amino acid substitution(s) tothe same cell-expressed counter structure ligand. The functionalactivity of the variant Fc-fusion molecules to modulate the activity ofT cells also is shown based on the calculated levels of IFN-gamma inculture supernatants (pg/ml) generated either i) with the indicatedvariant ECD-Fc fusion molecule coimmoblized with anti-CD3 or ii) withthe indicated variant ECD-Fc fusion molecule in an MLR assay. The Tablesalso depict the ratio of IFN-gamma produced by each variant ECD-Fccompared to the corresponding unmodified ECD-Fc in both functionalassays.

As shown, the selections resulted in the identification of a number ofCD80 or ICOSL IgSF domain variants that were affinity-modified toexhibit increased binding for at least one, and in some cases more thanone, cognate counter structure ligand. In addition, the results showedthat affinity modification of the variant molecules also exhibitedimproved activities to both increase and decrease immunological activitydepending on the format of the molecule. For example, coimmobilizationof the ligand likely provides a multivalent interaction with the cell tocluster or increase the avidity to favor agonist activity and increase Tcell activation compared to the unmodified (e.g. wildtype) ECD-Fcmolecule not containing the amino acid replacement(s). However, when themolecule is provided as a bivalent Fc molecule in solution, the sameIgSF domain variants exhibited an antagonist activity to decrease T cellactivation compared to the unmodified (e.g. wildtype) ECD-Fv moleculenot containing the amino acid replacement(s).

TABLE 6 CD80 variants selected against CD28. Molecule sequences, bindingdata, and costimulatory bioactivity data. Coimmobili- zation withBinding anti-CD3 MLR CD28 CTLA-4 PD-L1 IFN-gamma IFN-gamma SEQ ID MFIMFI MFI pg/ml levels pg/ml NO (parental (parental (parental (parental(parental CD80 mutation(s) (ECD) ratio) ratio) ratio) ratio) ratio)L70Q/A91G 55 125 283 6 93 716 (1.31) (1.36) (0.08) (1.12) (0.83)L70Q/A91G/T130A 56 96 234 7 99 752 (1.01) (1.13) (0.10) (1.19) (0.87)L70Q/A91G/I118A/ 57 123 226 7 86 741 T120S/T130A (1.29) (1.09) (0.10)(1.03) (0.86) V4M/L70Q/A91G/ 58 89 263 6 139 991 T120S/T130A (0.94)(1.26) (0.09) (1.67) (1.14) L70Q/A91G/T120S/ 59 106 263 6 104 741 T130A(1.12) (1.26) (0.09) (1.25) (0.86) V20L/L70Q/A91S/ 60 105 200 9 195 710T120S/T130A (1.11) (0.96) (0.13) (2.34) (0.82) S44P/L70Q/A91G/ 61 88 1345 142 854 T130A (0.92) (0.64) (0.07) (1.71) (0.99) L70Q/A91G/E117G/ 62120 193 6 98 736 T120S/T130A (1.27) (0.93) (0.08) (1.05) (0.85)A91G/T120S/ 63 84 231 44 276 714 T130A (0.89) (1.11) (0.62) (3.33)(0.82) L70R/A91G/T120S/ 64 125 227 6 105 702 T130A (1.32) (1.09) (0.09)(1.26) (0.81) L70Q/E81A/A91G/ 65 140 185 18 98 772 T120S/I127T/ (1.48)(0.89) (0.25) (1.18) (0.89) T130A L70Q/Y87N/A91G/ 66 108 181 6 136 769T130A (1.13) (0.87) (0.08) (1.63) (0.89) T28S/L70Q/A91G/ 67 32 65 6 120834 E95K/T120S/T130A (0.34) (0.31) (0.08) (1.44) (0.96) N63S/L70Q/A91G/68 124 165 6 116 705 T120S/T130A (1.30) (0.79) (0.08) (1.39) (0.81)K36E/I67T/L70Q/ 69 8 21 5 53 852 A91G/T120S/ (0.09) (0.10) (0.08) (0.63)(0.98) T130A/N152T E52G/L70Q/A91G/ 70 113 245 6 94 874 T120S/T130A(1.19) (1.18) (0.08) (1.13) (1.01) K37E/F59S/L70Q/ 71 20 74 6 109 863A91G/T120S/ (0.21) (0.36) (0.08) (1.31) (1.00) T130A A91G/S103P 72 39 569 124 670 (0.41) (0.27) (0.13) (1.49) (0.77) K89E/T130A 73 90 148 75 204761 (0.95) (0.71) (1.07) (2.45) (0.88) A91G 74 96 200 85 220 877 (1.01)(0.96) (1.21) (2.65) (1.01) D60V/A91G/T120S/ 75 111 222 12 120 744 T130A(1.17) (1.07) (0.18) (1.44) (0.86) K54M/A91G/T120S 76 68 131 5 152 685(0.71) (0.63) (0.08) (1.83) (0.79) M38T/L70Q/E77G/ 77 61 102 5 119 796A91G/T120S/ (0.64) (0.49) (0.07) (1.43) (0.92) T130A/N152TR29H/E52G/L70R/ 78 100 119 5 200 740 E88G/A91G/T130A (1.05) (0.57)(0.08) (2.41) (0.85) Y31H/T41G/L70Q/ 79 85 85 6 288 782 A91G/T120S/(0.89) (0.41) (0.08) (3.47) (0.90) T130A V68A/110A 80 103 233 48 163 861(1.08) (1.12) (0.68) (1,96) (0.99) S66H/D90G/T110A/ 81 33 121 11 129 758F116L (0.35) (0.58) (0.15) (1.55) (0.88) R29H/E52G/T120S/ 82 66 141 11124 800 T130A (0.69) (0.68) (0.15) (1.49) (0.92) A91G/L102S 83 6 6 5 75698 (0.06) (0.03) (0.08) (0.90) (0.81) I67T/L70Q/A91G/ 84 98 160 5 1751794 T120S (1.03) (0.77) (0.08) (21.1) (0.92) L70Q/A91G/T110A/ 85 8 14 577 656 T120S/T130A (0.09) (0.07) (0.07) (0.93) (0.76) M38V/T41D/M43I/ 865 8 8 82 671 W50G/D76G/V83A/ (0.06) (0.04) (0.11) (0.99) (0.78)K89E/T120S/T130A V22A/L70Q/S121P 87 5 7 5 105 976 (0.06) (0.04) (0.07)(1.27) (1.13) A12V/S15F/Y31H/ 88 6 6 5 104 711 T41G/T130A/P137L/ (0.06)(0.03) (0.08) (1.25) (0.82) N152T I67F/L70R/E88G/ 89 5 6 6 62 1003A91G/T120S/T130A (0.05) (0.03) (0.08) (0.74) (1.16) E24G/L25P/L70Q/ 9026 38 8 101 969 T120S (0.27) (0.18) (0.11) (1.21) (1.12)A91G/F92L/F108L/ 91 50 128 16 59 665 T120S (0.53) (0.61) (0.11) (0.71)(0.77) WT CD80 28 95 208 70 83 866 (1.00) (1.00) (1.00) (1.00) (1.00)

TABLE 7 CD80 variants selected against PD-Li. Molecule sequences,binding data, and costimulatory bioactivity data. CoimmobilizationBinding with anti-CD3 MLR SEQ CD28 CTLA-4 PD-L1 IFN-gamma IFN-gamma IDMFI MFI MFI pg/ml levels pg/ml NO (parental (parental (parental(parental (parental CD80 mutation(s) (ECD) ratio) ratio) ratio) ratio)ratio) R29D/Y31L/Q33H/ 92 1071 1089 37245 387 5028 K36G/M38I/T41A/(0.08) (0.02) (2.09) (0.76) (0.26) M43R/M47T/E81V/ L85R/K89N/A91T/F92P/K93V/R94L/ I118T/N149S R29D/Y31L/Q33H/ 93 1065 956 30713 400 7943K36G/M38I/T41A/ (0.08) (0.02) (1.72) (0.79) (0.41) M43R/M47T/E81V/L85R/K89N/A91T/ F92P/K93V/R94L/ N144S/N149S R29D/Y31L/Q33H/ 94 926 95447072 464 17387 K36G/M38I/T41A/ (0.07) (0.02) (2.64) (0.91) (0.91)M42T/M43R/M47T/ E81V/L85R/K89N/ A91T/F92P/K93V/ R94L/L148S/N149SE24G/R29D/Y31L/ 95 1074 1022 1121 406 13146 Q33H/K36G/M38I/ (0.08)(0.02) (0.06) (0.80) (0.69) T41A/M43R/M47T/ F59L/E81V/L85R/K89N/A91T/F92P/ K93V/R94L/H96R R29D/Y31L/Q33H/ 96 1018 974 25434 40524029 K36G/M38I/T41A/ (0.08) (0.02) (1.43) (0.80) (1.25) M43R/M47T/E81V/L85R/K89N/A91T/ F92P/K93V/R94L/ N149S R29V/M43Q/E81R/ 97 1029 996 1575342 11695 L85I/K89R/D9OL/ (0.08) (0.02) (0.09) (0.67) (0.61)A91E/F92N/K93Q/ R94G T41I/A91G 98 17890 50624 12562 433 26052 (1.35)(1.01) (0.70) (0.85) (1.36) K89R/D90K/A91G/ 99 41687 49429 20140 7736345 F92Y/K93R/N122S/ (3.15) (0.99) (1.13) (1.52) (0.33) N178SK89R/D90K/A91G/ 100 51663 72214 26405 1125 9356 F92Y/K93R (3.91) (1.44)(1.48) (2.21) (0.49) K36G/K37Q/M38I/ 101 1298 1271 3126 507 3095F59L/E81V/L85R/ (0.10) (0.03) (0.18) (1.00) (0.16) K89N/A91T/F92P/K93V/R94L/E99G/ T130A/N149S AE88D/K89R/D90K/ 102 31535 50868 29077 9445922 A91G/F92Y/K93R (2.38) (1.02) (1.63) (1.85) (0.31) K36G/K37Q/M38I/103 1170 1405 959 427 811 L40M (0.09) (0.03) (0.05) (0.84) (0.04) K36G104 29766 58889 20143 699 30558 (2.25) (1.18) (1.13) (1.37) (1.59)WTCD80 28 13224 50101 17846 509 19211 (1.00) (1.00) (1.00) (1.00) (1.00)

TABLE 8 ICOSL variants selected against CD28 or ICOS. Moleculesequences, binding data, and costimulatory bioactivity data.Coimmobilization with anti-CD3 MLR Binding IFN-gamma IFN-gamma SEQ ICOSOD CD28 MFI pg/ml levels pg/ml ICOSL ID NO (parental (parental (parental(parental mutation(s) (ECD) ratio) ratio) ratio) ratio) N52S 109 1.33162 1334  300 (1.55) (9.00) (1.93)  (0.44) N52H 110 1.30 368 1268   39(1.51) (20.44) (1.83)  (0.06) N52D 111 1.59 130 1943  190 (1.85) (7.22)(2.80)  (0.28) N52Y/N57Y/ 112 1.02 398 510*  18 F138L/L203P (1.19)(22.11) (1.47*) (0.03) N52H/N57Y/Q100P 113 1.57 447 2199   25 (1.83)(24.83) (3.18)  (0.04) N52S/Y146C/ 114 1.26 39 1647  152 Y152C (1.47)(2.17) (2.38)  (0.22) N52H/C198R 115 1.16 363 744* ND (1.35) (20.17)(2.15*) (ND) N52H/C140D/T225A 116 ND 154 522* ND (ND) (8.56) (1.51*)(ND) N52H/C198R/T225A 117 1.41 344 778*  0 (1.64) (19.11) (2.25*) (0)  N52H/K92R 118 1.48 347 288*  89 (1.72) (19.28) (0.83*) (0.13) N52H/S99G119 0.09 29 184* 421 (0.10) (1.61) (0.53*) (0.61) N52Y 120 0.08 18 184*568 (0.09) (1.00) (0.53*) (0.83) N57Y 121 1.40 101 580* 176 (1.63)(5.61) (1.68*) (0.26) N57Y/Q100P 122 0.62 285 301* 177 (0.72) (15.83)(0.87*) (0.26) N52S/S130G/ 123 0.16 24 266* 1617  Y152C (0.19) (1.33)(0.77*) (2.35) N52S/Y152C 124 0.18 29 238* 363 (0.21) (1.61) (0.69*)(0.53) N52S/C198R 125 1.80 82 1427  201 (2.09) (4.56) (2.06)  (0.29)N52Y/N57Y/Y152C 126 0.08 56 377* 439 (0.09) (3.11) (1.09*) (0.64)N52Y/N57Y/ 127 ND 449 1192  ND H129P/C198R (ND) (24.94) (1.72)  (ND)N52H/L161P/ 128 0.18 343 643* 447 C198R (0.21) (19.05) (1.86*) (0.65)N52S/T113E 129 1.51 54 451* 345 (1.76) (3.00) (1.30*) (0.50) S54A 1301.62 48 386* 771 (1.88) (2.67) (1.12*) (1.12) N52D/S54P 131 1.50 38 476*227 (1.74) (2.11) (1.38*) (0.33) N52K/L208P 132 1.91 291 1509  137(2.22) (16.17) (2.18)  (0.20) N52S/Y152H 133 0.85 68 2158  221 (0.99)(3.78) (3.12)  (0.32) N52D/V151A 134 0.90 19 341* 450 (1.05) (1.06)(0.99*) (0.66) N52H/I143T 135 1.83 350 2216  112 (2.13) (19.44) (3.20) (0.16) N52S/L80P 136 0.09 22 192* 340 (0.10) (1.22) (0.55*) (0.49)F120S/Y152H/N201S 137 0.63 16 351* 712 (0.73) (0.89) (1.01*) (1.04)N52S/R75Q/L203P 138 1.71 12 1996  136 (1.99) (0.67) (2.88)  (0.20)N52S/D158G 139 1.33 39 325* 277 (1.55) (2.17) (0.94*) (0.40) N52D/Q133H140 1.53 104 365* 178 (1.78) (5.78) (1.05*) (0.26) WT ICOSL 32 0.86 18692/346* 687 (1.00) (1.00) (1.00)  (1.00) *Parental ratio calculatedusing 346 pg/ml IFN-gamma for WT ICOSL

Example 7 Ligand Binding Competition Assay

As shown in Example 6, several CD80 variant molecules exhibited improvedbinding to one or both of CD28 and PD-L1. To further assess the bindingactivity of CD80 to ligands CD28 and PD-L1, this Example describes aligand competition assay assessing the non-competitive nature ofexemplary CD80 variants to bind both CD28 and PD-L1.

An ELISA based binding assay was set up incorporating plate-bound CD80variant A91G ECD-Fc to assess the ability of CD80 to simultaneously bindCD28 and PD-L1. Maxisorp 96 well ELISA plates (Nunc, USA) were coatedovernight with 100 nM human recombinant CD80 variant A91G ECD-Fc fusionprotein in PBS. The following day unbound protein was washed out, andthe plate was blocked with 1% bovine serum albumin (Millipore, USA)/PBSfor 1 hour at room temperature. This blocking reagent was washed outthree times with PBS/0.05% Tween, which included a two minute incubationon a platform shaker for each wash.

In one arm of the competition assay, CD80 was incubated with CD28, andthen CD28-bound CD80 was then assessed for competitive binding in thepresence of either the other known CD80 ligand counter structures PD-L1or CTLA-4 or negative control ligand PD-L2. Specifically, biotinylatedrecombinant human CD28 Fc fusion protein (rCD28.Fc; R&D Systems) wastitrated into the wells starting at 10 nM, diluting out for eight pointswith 1:2 dilutions in 25 μl volume. Immediately after adding thebiotinylated rCD28.Fc, unlabeled competitive binders, recombinant humanPD-L1 monomeric his-tagged protein, recombinant human CTLA-4 monomerichis-tagged protein, or a negative control human recombinant PD-L2 Fcfusion protein (R&D Systems) were added to wells at 2000/1000/500 nMrespectively in 25 μl volume for a final volume of 50 μl. These proteinswere incubated together for one hour before repeating the three washsteps as described above.

After washing, 2.5 ng per well of HRP-conjugated streptavidin (JacksonImmunoresearch, USA) was diluted in 1% BSA/PBS and added to wells todetect bound biotinylated rCD28.Fc. After one hour incubation, wellswere washed again three times as described above. To detect signal, 50μl of TMB substrate (Pierce, USA) was added to wells following wash andincubated for 7 minutes, before adding 50 ul 2M sulfuric acid stopsolution. Optical density was determined on an Emax Plus microplatereader (Molecular Devices, USA). Optical density values were graphed inPrism (Graphpad, USA).

The results are set forth in FIG. 1A. The results showed decreasedbinding of biotinylated rCD28.Fc to the CD80 variant A91G ECD-Fc fusionprotein with titration of the rCD28.Fc. When rCD28.Fc binding wasperformed in the presence of non-competitive control protein, rPDL2,there was no decrease in CD28 binding for CD80 (solid triangle). Incontrast, a competitive control protein, rCTLA-4, when incubated withthe CD28.Fc, did result in decreased CD28 binding for CD80 as expected(x line). When recombinant PD-L1 was incubated with CD28.Fc, no decreasein CD28 binding to CD80 was observed, which demonstrated that theepitopes of CD28 and PD-L1 for CD80 are non-competitive. Binding of therecombinant PD-L1 protein used in the CD28 competition assay to CD80 wasconfirmed by incubating the biotinylated PD-L1 in the presence ofnon-biotinylated rCD28.Fc (square).

The reverse competition also was set up in which CD80 was incubated withPD-L1, and then PD-L1-bound CD80 was then assessed for competitivebinding in the presence of either the other known CD80 ligand counterstructures CD28 or CTLA-4 or negative control ligand PD-L2.Specifically, the assay was performed by titrating biotinylatedrecombinant human PD-L1-his monomeric protein into wells containing therecombinant CD80 variant. Because binding is weaker with this ligand,titrations started at 5000 nM with similar 1:2 dilutions over eightpoints in 25 μL. When the rPD-L1-his was used to detect binding, thecompetitive ligands human rCD28.Fc, human rCTLA-4.Fc, or human rPD-L2.Fccontrol were added at 2.5 nM final concentration in 25 μl for a totalvolume of 50 μl. The subsequent washes, detection, and OD measurementswere the same as described above.

The results are set forth in FIG. 1B. Titrated PD-L1-his binding aloneconfirmed that PD-L1 bound to the CD80 variant A91G ECD-Fc fusionmolecule immobilized on the plate (square). When PD-L1-his binding wasperformed in the presence of non-competitive control protein, rPDL2,there was no decrease in PD-L1 binding for CD80 (triangle). TheCD28-competitive control protein, rCTLA-4, when incubated with thePD-L1-his, did not result in decreased PD-L1 binding for CD80 (x line),even though CTLA-4 is competitive for CD28. This result furtherdemonstrated that lack of competition between CD28 and PD-L1 for CD80binding. Finally, when PD-L1-his was incubated with CD28.Fc, no decreasein PD-L1 binding to CD80 was observed, which demonstrated that theepitopes of CD28 and PD-L1 for CD80 are non-competitive.

Thus, the results showed that CTLA-4, but not PD-L1 or the negativecontrol PD-L2, competed for binding of CD28 to CD80 (FIG. 1A) and thatCD28, CTLA-4, and PD-L2 did not compete for binding of PD-L1 to CD80(FIG. 1B). Thus, these results demonstrated that CD28 and PD-L1 arenon-competitive binders of CD80, and that this non-competitive bindingcan be demonstrated independently of which ligand is being detected inthe ELISA.

Example 8 Generation and Assessment of Stacked Molecules ContainingDifferent Affinity-Modified Domains

Selected variant molecules described above that were affinity-modifiedfor one or more counter structure ligand were used to generate “stack”molecule (i.e., Type II immunomodulatory protein) containing two or moreaffinity-modified IgSF domains. Stack constructs were obtained asgeneblocks (Integrated DNA Technologies, Coralville, Iowa) that encodethe stack in a format that enables its fusion to Fc by standard Gibsonassembly using a Gibson assembly kit (New England Biolabs).

The encoding nucleic acid molecule of all stacks was generated to encodea protein designed as follows: Signal peptide, followed by the firstvariant IgV of interest, followed by a 15 amino acid linker which iscomposed of three GGGGS(G4S) motifs (SEQ ID NO:228), followed by thesecond IgV of interest, followed by two GGGGS linkers (SEQ ID NO: 229)followed by three alanines (AAA), followed by a human IgG1 Fc asdescribed above. To maximize the chance for correct folding of the IgVdomains in each stack, the first IgV was preceded by all residues thatnormally occur in the wild-type protein between this IgV and the signalpeptide (leading sequence). Similarly, the first IgV was followed by allresidues that normally connect it in the wild-type protein to either thenext Ig domain (typically an IgC domain) or if such a second IgV domainis absent, the residues that connect it to the transmembrane domain(trailing sequence). The same design principle was applied to the secondIgV domain except that when both IgV domains were derived from sameparental protein (e.g. a CD80 IgV stacked with another CD80 IgV), thelinker between both was not duplicated.

Table 9 sets forth the design for exemplary stacked constructs. Theexemplary stack molecules shown in Table 9 contain the IgV domains asindicated and additionally leading or trailing sequences as describedabove. In the Table, the following components are present in order:signal peptide (SP; SEQ ID NO:225), IgV domain 1 (IgV1), trailingsequence 1 (TS1), linker 1 (LR1; SEQ ID NO:228), IgV domain 2 (IgV2),trailing sequence 2 (TS2), linker 2 (LR2; SEQ ID NO:230) and Fc domain(SEQ ID NO:226 containing C5S/N82G amino acid substitution). In somecases, a leading sequence 1 (LS1) is present between the signal peptideand IgV1 and in some cases a leading sequence 2 (LS2) is present betweenthe linker and IgV2.

TABLE 9 Amino acid sequence (SEQ ID NO) of components of exemplarystacked constructs First domain Second domain SP LS1 IgV1 TS1 LR1 LS2IgV2 TS2 LR2 Fc NKp30 WT + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + ICOSLWT NO: 214 NO: 235 NO: 196 NO: 233 NKp30 + − SEQ ID SEQ ID + − SEQ IDSEQ ID + + L30V/A60V/S64P/ NO: 215 NO: 235 NO: 212 NO: 233 S86G ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R NKp30 + − SEQ ID SEQ ID + − SEQ ID SEQID + + L30V/A60V/S64P/ NO: 215 NO: 235 NO: 199 NO: 233 S86G) ICOSL N52DNKp30 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + L30V/A60V/S64P/ NO: 215NO: 235 NO: 201 NO: 233 S86G ICOSL N52H/N57Y/Q100P ICOSL WT + − SEQ IDSEQ ID + − SEQ ID SEQ ID + + Nkp30 WT NO: 196 NO: 233 NO: 214 NO: 235ICOSL N52D + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + NKp30 NO: 199 NO: 233NO: 215 NO: 235 L30V/A60V/S64P/ S86G ICOSL + − SEQ ID SEQ ID + − SEQ IDSEQ ID + + N52H/N57Y/Q100P NO: 201 NO: 233 NO: 215 NO: 235 NKp30L30V/A60V/S64P/ S86G Domain 1: NKp30 + − SEQ ID SEQ ID + − SEQ ID SEQID + + WT NO: 214 NO: 235 NO: 152 NO: 231 Domain 2: CD80 WT Domain 1:NKp30 + − SEQ ID SEQ ID + SEQ ID SEQ ID SEQ ID + + WT NO: 214 NO: 235NO: 236 NO: 220 NO: 237 Domain 2: CD86 WT Domain 1: NKp30 + − SEQ ID SEQID + − SEQ ID SEQ ID + + L30V/A60V/S64P/ NO: 215 NO: 235 NO: 192 NO: 231S86G Domain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109SDomain 1: NKp30 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + L30V/A60V/S64P/NO: 215 NO: 235 NO: 175 NO: 231 S86G Domain 2: CD80 I67T/L70Q/A91G/T120S Domain 1: NKp30 + − SEQ ID SEQ ID + SEQ ID SEQ ID SEQ ID + +L30V/A60V/S64P/ NO: 215 NO: 235 NO: 236 NO: 221 NO: 237 S86G Domain 2:CD86 Q35H/H90L/Q102H Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQID + + WT NO: 152 NO: 231 NO: 214 NO: 235 Domain 2: Nkp30 WT Domain 1:CD86 + SEQ ID SEQ ID SEQ ID + − SEQ ID SEQ ID + + WT NO: 236 NO: 220 NO:237 NO: 214 NO: 235 Domain 2: Nkp30 WT Domain 1: CD80 + − SEQ ID SEQID + − SEQ ID SEQ ID + + R29H/Y31H/T41G/ NO: 192 NO: 231 NO: 215 NO: 235Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2: NKp30 L30V/A60V/S64P/ S86GDomain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + I67T/L70Q/A91G/NO: 175 NO: 231 NO: 215 NO: 235 T120S Domain 2: NKp30 L30V/A60V/S64P/S86G Domain 1: CD86 + SEQ ID SEQ ID SEQ ID + − SEQ ID SEQ ID + +Q35H/H90L/Q102H NO: 236 NO: 221 NO: 237 NO: 215 NO: 235 Domain 2: NKp30L30V/A60V/S64P/ S86G Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQID + + WT NO: 152 NO: 231 NO: 196 NO: 233 Domain 2: ICOSL WT Domain 1:CD80 + − SEQ ID SEQ ID + SEQ ID SEQ ID SEQ ID + + WT NO: 152 NO: 231 NO:236 NO: 220 NO: 237 Domain 2: CD86 WT Domain 1: CD80 + − SEQ ID SEQ ID +− SEQ ID SEQ ID + + WT NO: 152 NO: 231 NO: 152 NO: 231 Domain 2: CD80 WTDomain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + E88D/K89R/D90K/NO: 189 NO: 231 NO: 192 NO: 231 A91G/F92Y/K93R Domain 2: CD80R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1: CD80 + − SEQID SEQ ID + − SEQ ID SEQ ID + + A12T/H18L/N43V/ NO: 193 NO: 231 NO: 192NO: 231 F59L/E77K/P109S/ I118T Domain 2: CD80 R29H/Y31H/T41G/Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1: CD80 + − SEQ ID SEQ ID + − SEQID SEQ ID + + A12T/H18L/N43V/ NO: 193 NO: 231 NO: 175 NO: 231F59L/E77K/P109S/ I118T Domain 2: CD80 I67T/L70Q/A91G/ T120S Domain 1:CD80 + − SEQ ID SEQ ID + SEQ ID SEQ ID SEQ ID + + E88D/K89R/D90K/ NO:189 NO: 231 NO: 236 NO: 221 NO: 237 A91G/F92Y/K93R Domain 2: CD86Q35H/H90L/Q102H Domain 1: CD80 + − SEQ ID SEQ ID + SEQ ID SEQ ID SEQID + + A12T/H18L/N43V/ NO: 193 NO: 231 NO: 236 NO: 221 NO: 237F59L/E77K/P109S/ I118T Domain 2: CD86 Q35H/H90L/Q102H Domain 1: CD80 + −SEQ ID SEQ ID + − SEQ ID SEQ ID + + E88D/K89R/D90K/ NO: 189 NO: 231 NO:213 NO: 233 A91G/F92Y/K93R Domain 2: ICOSL N525/N57Y/H94D/L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ IDSEQ ID + + A12T/H18L/N43V/ NO: 193 NO: 231 NO: 213 NO: 233F59L/E77K/P109S/ I118T Domain 2: ICOSL N52S/N57Y/H94D/ L96F/L98F/Q100R/G103E/F120S Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + +A12T/H18L/N43V/ NO: 193 NO: 231 NO: 199 NO: 233 F59L/E77K/P109S/ I118TDomain 2: ICOSL N52D Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQID + + E88D/K89R/D90K/ NO: 189 NO: 231 NO: 201 NO: 233 A91G/F92Y/K93RDomain 2: ICOSL N52H/N57Y/Q100P Domain 1: CD80 + − SEQ ID SEQ ID + − SEQID SEQ ID + + A12T/H18L/N43V/ NO: 193 NO: 231 NO: 201 NO: 233F59L/E77K/P109S/ I118T Domain 2: ICOSL N52H/N57Y/Q100P Domain 1: ICOSL +− SEQ ID SEQ ID + − SEQ ID SEQ ID + + WT NO: 196 NO: 233 NO: 152 NO: 231Domain 2: CD80 WT Domain 1: CD86 + SEQ ID SEQ ID SEQ ID + − SEQ ID SEQID + + WT NO: 236 NO: 220 NO: 237 NO: 152 NO: 231 Domain 2: CD80 WTDomain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + R29H/Y31H/T41G/NO: 192 NO: 231 NO: 189 NO: 231 Y87N/E88G/K89E/ D90N/A91G/P109S Domain2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: CD80 + − SEQ ID SEQID + − SEQ ID SEQ ID + + R29H/Y31H/T41G/ NO: 192 NO: 231 NO: 193 NO: 231Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2: CD80 A12T/H18L/N43V/F59L/E77K/P109S/ I118T Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQID + + I67T/L70Q/A91G/ NO: 175 NO: 231 NO: 189 NO: 231 T120S Domain 2:CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: CD80 + − SEQ ID SEQ ID + −SEQ ID SEQ ID + + I67T/L70Q/A91G/ NO: 175 NO: 231 NO: 193 NO: 231 T120SDomain 2: CD80 A12T/H18L/N43V/ F59L/E77K/P109S/ I118T Domain 1: CD86 +SEQ ID SEQ ID SEQ ID + − SEQ ID SEQ ID + + Q35H/H90L/Q102H NO: 236 NO:221 NO: 237 NO: 189 NO: 231 Domain 2: CD80 E88D/K89R/D90K/A91G/F92Y/K93R Domain 1: CD86 + SEQ ID SEQ ID SEQ ID + − SEQ ID SEQID + + Q35H/H90L/Q102H NO: 236 NO: 221 NO: 237 NO: 193 NO: 231 Domain 2:CD80 A12T/H18L/N43V/ F59L/E77K/P109S/ I118T Domain 1: ICOSL + − SEQ IDSEQ ID + − SEQ ID SEQ ID + + N525/N57Y/H94D/ NO: 213 NO: 233 NO: 189 NO:231 L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80 E88D/K89R/D90K/A91G/F92Y/K93R Domain 1: ICOSL + − SEQ ID SEQ ID + − SEQ ID SEQ ID + +N52S/N57Y/H94D/ NO: 213 NO: 233 NO: 193 NO: 231 L96F/L98F/Q100R/G103E/F120S Domain 2: CD80 A12T/H18L/N43V/ F59L/E77K/P109S/ I118T Domain1: ICOSL + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + N52D NO: 199 NO: 233NO: 189 NO: 231 Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:ICOSL + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + N52D NO: 199 NO: 233 NO:193 NO: 231 Domain 2: CD80 A12T/H18L/N43V/ F59L/E77K/P109S/ I118T Domain1: ICOSL + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + N52H/N57Y/Q100P NO: 201NO: 233 NO: 189 NO: 231 Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93RDomain 1: ICOSL + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + N52H/N57Y/Q100PNO: 201 NO: 233 NO: 193 NO: 231 Domain 2: CD80 A12T/H18L/N43V/F59L/E77K/P109S/ I118T Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQID + + V68M/L70P/L72P/ NO: 195 NO: 231 NO: 189 NO: 231 K86E Domain 2:CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: CD80 + − SEQ ID SEQ ID + −SEQ ID SEQ ID + + R29V/Y31F/K36G/ NO: 194 NO: 231 NO: 189 NO: 231M38L/N43Q/E81R/ V83I/L85I/K89R/ D90L/A91E/F92N/ K93Q/R94G Domain 2: CD80E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: CD80 + − SEQ ID SEQ ID + − SEQID SEQ ID + + V68M/L70P/L72P/ NO: 195 NO: 231 NO: 193 NO: 231 K86EDomain 2: CD80 A12T/H18L/N43V/ F59L/E77K/P109S/ I118T Domain 1: CD80 + −SEQ ID SEQ ID + − SEQ ID SEQ ID + + R29V/Y31F/K36G/ NO: 194 NO: 231 NO:193 NO: 231 M38L/N43Q/E81R/ V83I/L85I/K89R/ D90L/A91E/F92N/ K93Q/R94GDomain 2: CD80 A12T/H18L/N43V/ F59L/E77K/P109S/ I118T Domain 1: CD80 + −SEQ ID SEQ ID + − SEQ ID SEQ ID + + E88D/K89R/D90K/ NO: 189 NO: 231 NO:195 NO: 231 A91G/F92Y/K93R Domain 2: CD80 V68M/L70P/L72P/ K86E Domain 1:CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + E88D/K89R/D90K/ NO: 189 NO:231 NO: 194 NO: 231 A91G/F92Y/K93R Domain 2: CD80 R29V/Y31F/K36G/M38L/N43Q/E81R/ V83I/L85I/K89R/ D90L/A91E/F92N/ K93Q/R94G Domain 1:CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + A12T/H18L/N43V/ NO: 193 NO:231 NO: 195 NO: 231 F59L/E77K/P109S/ I118T Domain 2: CD80V68M/L70P/L72P/ K86E Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQID + + A12T/H18L/N43V/ NO: 193 NO: 231 NO: 194 NO: 231 F59L/E77K/P109S/I118T Domain 2: CD80 R29V/Y31F/K36G/ M38L/N43Q/E81R/ V83I/L851/K89R/D90L/A91E/F92N/ K93Q/R94G Domain 1: CD86 + SEQ ID SEQ ID SEQ ID + − SEQID SEQ ID + + WT NO: 236 NO: 220 NO: 237 NO: 196 NO: 233 Domain 2: ICOSLWT Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + +R29H/Y31H/T41G/ NO: 192 NO: 231 NO: 213 NO: 233 Y87N/E88G/K89E/D90N/A91G/P109S Domain 2: ICOSL N52S/N57Y/H94D/ L96F/L98F/Q100R/G103E/F120S Domain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + +I67T/L70Q/A91G/ NO: 175 NO: 231 NO: 213 NO: 233 T120S Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + − SEQ IDSEQ ID + − SEQ ID SEQ ID + + R29H/Y31H/T41G/ NO: 192 NO: 231 NO: 199 NO:233 Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2: ICOSL N52D Domain 1:CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + I67T/L70Q/A91G/ NO: 175 NO:231 NO: 199 NO: 233 T120S Domain 2: ICOSL N52D Domain 1: CD80 + − SEQ IDSEQ ID + − SEQ ID SEQ ID + + R29H/Y31H/T41G/ NO: 192 NO: 231 NO: 201 NO:233 Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2: ICOSL N52H/N57Y/Q100PDomain 1: CD80 + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + I67T/L70Q/A91G/NO: 175 NO: 231 NO: 201 NO: 233 T120S Domain 2: ICOSL N52H/N57Y/Q100PDomain 1: CD86 + SEQ ID SEQ ID SEQ ID + − SEQ ID SEQ ID + +Q35H/H90L/Q102H NO: 236 NO: 221 NO: 237 NO: 213 NO: 233 Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103EF120S Domain 1: CD86 + SEQ ID SEQID SEQ ID + − SEQ ID SEQ ID + + Q35H/H90L/Q102H NO: 236 NO: 221 NO: 237NO: 199 NO: 233 Domain 2: ICOSL N52D Domain 1: CD86 + SEQ ID SEQ ID SEQID + − SEQ ID SEQ ID + + Q35H/H90L/Q102H NO: 236 NO: 221 NO: 237 NO: 201NO: 233 Domain 2: ICOSL N52H/N57Y/Q100P Domain 1: ICOSL + − SEQ ID SEQID + SEQ ID SEQ ID SEQ ID + + WT NO: 196 NO: 233 NO: 236 NO: 220 NO: 237Domain 2: CD86 WT Domain 1: ICOSL + − SEQ ID SEQ ID + − SEQ ID SEQID + + N52S/N57Y/H94D/ NO: 213 NO: 233 NO: 192 NO: 231 L96F/L98F/Q100R/G103E/F120S Domain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/D90N/A91G/P109S Domain 1: ICOSL + − SEQ ID SEQ ID + − SEQ ID SEQ ID + +N52S/N57Y/H94D/ NO: 213 NO: 233 NO: 175 NO: 231 L96F/L98F/Q100R/G103E/F120S Domain 2: CD80 I67T/L70Q/A91G/ T120S Domain 1: ICOSL + − SEQID SEQ ID + − SEQ ID SEQ ID + + N52D NO: 199 NO: 233 NO: 192 NO: 231Domain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1:ICOSL + − SEQ ID SEQ ID + − SEQ ID SEQ ID + + N52D NO: 199 NO: 233 NO:175 NO: 231 Domain 2: CD80 I67T/L70Q/A91G/ T120S Domain 1: ICOSL + − SEQID SEQ ID + − SEQ ID SEQ ID + + N52H/N57Y/Q100P NO: 201 NO: 233 NO: 192NO: 231 Domain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109SDomain 1: ICOSL + − SEQ ID SEQ ID + SEQ ID SEQ ID SEQ ID + +N52S/N57Y/H94D/ NO: 213 NO: 233 NO: 236 NO: 221 NO: 237 L96F/L98F/Q100R/G103E/F120S Domain 2: CD86 Q35H/H90L/Q102H Domain 1: ICOSL + − SEQ IDSEQ ID + SEQ ID SEQ ID SEQ ID + + N52D NO: 199 NO: 233 NO: 236 NO: 221NO: 237 Domain 2: CD86 Q35H/H90L/Q102H Domain 1: ICOSL + − SEQ ID SEQID + SEQ ID SEQ ID SEQ ID + + N52H/N57Y/Q100P NO: 201 NO: 233 NO: 236NO: 221 NO: 237 Domain 2: CD86 Q35H/H90L/Q102H

High throughput expression and purification of the variantIgV-stacked-Fc fusion molecules containing various combinations ofvariant IgV domains from CD80, CD86, ICOSL or Nkp30 containing at leastone affinity-modified IgV domain were generated as described in Example5. Binding of the variant IgV-stacked-Fc fusion molecules to respectivecounter structures and functional activity by anti-CD3 coimmobilizationassay also were assessed as described in Example 6. For example,costimulatory bioactivity of the stacked IgSF Fc fusion proteins wasdetermined in a similar immobilized anti-CD3 assay as above. In thiscase, 4 nM of anti-CD3 (OKT3, Biolegend, USA) was coimmobilized with 4nM to 120 nM of human rB7-H6.Fc (R&D Systems, USA) or human rPD-L1.Fc(R&D Systems, USA) overnight on tissue-culture treated 96 well plates(Corning, USA). The following day unbound protein was washed off withPBS and 100,000 purified pan T cells were added to each well in 100 ulEx-Vivo 15 media (Lonza, Switzerland). The stacked IgSF domains weresubsequently added at concentrations ranging from 8 nM to 40 nM in avolume of 100 ul for 200 ul volume total. Cells were cultured 3 daysbefore harvesting culture supernatants and measuring human IFN-gammalevels with Duoset ELISA kit (R&D Systems, USA) as mentioned above.

The results are set forth in Tables 10-14. Specifically, Table 10 setsforth binding and functional activity results for variant IgV-stacked-Fcfusion molecules containing an NKp30 IgV domain and an ICOSL IgV domain.Table 11 sets forth binding and functional activity results for variantIgV-stacked-Fv fusion molecules containing an Nkp30 IgV domain and aCD80 or CD86 IgV domain. Table 12 sets forth binding and functionalactivity results for variant IgV-stacked-Fc fusion molecules containinga variant CD80 IgV domain and a CD80, CD86 or ICOSL IgV domain. Table 13sets forth binding and functional activity results for variantIgV-stacked-Fc fusion molecules containing two variant CD80 IgV domains.Table 14 sets forth results for variant IgV-stacked Fc fusion moleculescontaining a variant CD80 or CD86 IgV domain and a variant ICOSL IgVdomain.

For each of Tables 10-14, Column 1 indicates the structural organizationand orientation of the stacked, affinity modified or wild-type (WT)domains beginning with the amino terminal (N terminal) domain, followedby the middle WT or affinity modified domain located before the Cterminal human IgG1 Fc domains. Column 2 sets forth the SEQ ID NOidentifier for the sequence of each IgV domain contained in a respective“stack” molecule. Column 3 shows the binding partners which theindicated affinity modified stacked domains from column 1 were selectedagainst.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each stack molecule to cellsengineered to express various counter structure ligands and the ratio ofthe MFI compared to the binding of the corresponding stack moleculecontaining unmodified IgV domains not containing the amino acidsubstitution(s) to the same cell-expressed counter structure ligand. Thefunctional activity of the variant stack molecules to modulate theactivity of T cells also is shown based on the calculated levels ofIFN-gamma in culture supernatants (pg/ml) generated with the indicatedvariant stack molecule in solution and the appropriate ligandcoimmoblized with anti-CD3 as described in Example 6. The Tables alsodepict the ratio of IFN-gamma produced by each variant stack moleculecompared to the corresponding unmodified stack molecule in thecoimmobilization assay.

As shown, the results showed that it was possible to generate stackmolecules containing at least one variant IgSF domains that exhibitedaffinity-modified activity of increased binding for at least one cognatecounter structure ligand compared to a corresponding stack moleculecontaining the respective unmodified (e.g. wild-type) IgV domain. Insome cases, the stack molecule, either from one or a combination of bothvariant IgSF domains in the molecule, exhibited increased binding formore than one cognate counter structure ligand. The results also showedthat the order of the IgV domains in the stacked molecules could, insome cases, alter the degree of improved binding activity. In somecases, functional T cell activity also was altered when assessed in thetargeted coimmobilization assay.

TABLE 10 Stacked variant IgV Fc fusion proteins containing an NKp30 IgVdomain and an ICOS IgV domain Anti-CD3 Binding Activity coimmobilizationCounter B7H6 MFI ICOS MFI CD28 MFI assay pg/ml Domain Structure SEQ IDstructure (WT (WT (WT IFN-gamma N terminal to C terminal: NO selectedparental parental parental (WT parental domain 1/domain 2/Fc (IgV)against MFI ratio) MFI ratio) MFI ratio) IFN-gamma ratio) Domain 1:NKp30 WT 214 — 64538 26235 6337 235 Domain 2: ICOSL WT 196 (1.00) (1.00)(1.00) (1.00) Domain 1: NKp30 (L30V 215 B7-H6 59684 12762 9775 214 A60VS64P S86G) (0.92) (0.49) (1.54) (0.91) Domain 2: ICOSL (N52S 212 ICOS-N57Y H94D L96F L98F CD28 Q100R) Domain 1: NKp30 (L30V 215 B7-H6 6547030272 9505 219 A60V S64P S86G) (1.01) (1.15) (1.50) (0.93) Domain 2:ICOSL (N52D) 199 ICOS- CD28 Domain 1: NKp30 (L30V 215 B7-H6 38153 2790311300 189 A60V S64P S86G)/ (0.59) (1.06) (1.78) (0.80) Domain 2: ICOSL(N52H 201 ICOS- N57Y Q100P) CD28 Domain 1: ICOSL WT 196 — 117853 703207916 231 Domain 2: Nkp30 WT 214 (1.0) (1.0) (1.0) (1.0) Domain 1: ICOSL(N52D) 199 ICOS- 100396 83912 20778 228 CD28 (0.85) (1.19) (2.62) (0.98)Domain 2: NKp30 (L30V 215 B7-H6 A60V S64P S86G) Domain 1: ICOSL (N52H201 ICOS- 82792 68874 72269 561 N57Y Q100P) CD28 (0.70) (0.98) (9.12)(2.43) Domain 2: NKp30 (L30V 215 B7-H6 A60V S64P S86G)

TABLE 11 Stacked variant IgV Fc fusion proteins containing an NKp30 IgVdomain and a CD80 or CD86 IgV domain Anti-CD3 coimmobilization CounterBinding Activity assay Domain Structure SEQ structure B7H6 MFI CD28 MFIpg/ml IFN-gamma N terminal to C terminal: ID NO selected (WT parental(WT parental (WT parental IFN- domain 1/domain 2/Fc (IgV) against MFIratio) MFI ratio) gamma ratio) Domain 1: NKp30 WT 214 — 88823 7022 68Domain 2: CD80 WT 152 (1.00) (1.00) (1.00) Domain 1: NKp30 WT 214 —14052 1690 92 Domain 2: CD86 WT 220 (1.00) (1.00) (1.00) Domain 1: NKp30(L30V 215 B7-H6 53279 9027 94 A60V S64P S86G) (0.60) (1.29) (1.38)Domain 2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/A91G/ P109SDomain 1: NKp30 (L30V 215 B7-H6 41370 11240 60 A60V S64P S86G) (0.47)(1.60) (0.88) Domain 2: CD80 175 CD28 167T/L70Q/A91G/T120S Domain 1:NKp30 (L30V 215 B7-H6 68480 9115 110 A60V S64P S86G)/ (4.87) (5.39)(1.19) Domain 2: CD86 221 CD28 Q35H/H90L/Q102H Domain 1: CD80 WT 152 —110461 13654 288 Domain 2: Nkp30 WT 214 (1.00) (1.00) (1.00) Domain 1:CD86 WT 220 CD28 128899 26467 213 Domain 2: Nkp30 WT 214 B7-H6 (1.00)(1.00) (1.00) Domain 1: CD80 192 CD28 55727 4342 100R29H/Y31H/T41G/Y87N/ (0.50) (0.32) (0.35) E88G/K89E/D90N/A91G/ P109SDomain 2: NKp30 (L30V 215 B7-H6 A60V S64P S86G) Domain 1: CD80 175 CD2840412 7094 84 167T/L70Q/A91G/T120S (0.37) (0.52) (0.29) Domain 2: NKp30(L30V 215 B7-H6 A60V S64P S86G) Domain 1: CD86 221 CD28 220836 11590 113Q35H/H90L/Q102H (1.71) (0.44) (0.53) Domain 2: NKp30 (L30V 215 B7-H6A60V S64P S86G)

TABLE 12 Stacked variant IgV Fc fusion proteins containing a CD80 IgVdomain and a CD80, CD86, or ICOSL IgV domain Anti-CD3 Binding Activitycoimmobilization SEQ Counter CD28 MFI PD-L1 ICOS assay pg/ml DomainStructure ID structure (WT MFI (WT MFI (WT IFN-gamma N terminal to Cterminal: NO selected parental parental parental (WT parental domain1/domain 2/Fc (IgV) against MFI ratio) MFI ratio) MFI ratio) IFN-gammaratio) Domain 1: CD80 WT 152 1230 2657 11122 69 Domain 2: ICOSL WT 196(1.00) (1.00) (1.00) (1.00) Domain 1: CD80 WT 152 60278 2085 59 Domain2: CD86 WT 220 (1.00) (1.00) (1.00) Domain 1: CD80 WT 152 1634 6297 98Domain 2: CD80 WT 152 (1.00) (1.00) (1.00) Domain 1: CD80 189 PD-L1 43084234 214 E88D/K89R/D90K/A91G/ (2.64) (0.67) (2.18) F92Y/K93R Domain 2:CD80 192 CD28 R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/A91G/ P109S Domain 1:CD80 193 PD-L1 7613 2030 137 A12T/H18L/N43V/F59L/ (4.66) (0.32) (1.40)E77K/P109S/I118T Domain 2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/ P109S Domain 1: CD80 193 PD-L1 3851 3657 81A12T/H18L/N43V/F59L/ (2.36) (0.58) (0.83) E77K/P109S/I118T Domain 2:CD80 175 CD28 I67T/L70Q/A91G/T120S Domain 1: CD80 189 PD-L1 4117 2914 96E88D/K89R/D90K/A91G/ (0.07) (1.40) (1.63) F92Y/K93R Domain 2: CD86 221CD28 Q35H/H90L/Q102H Domain 1: CD80 193 PD-L1 2868 3611 94A12T/H18L/N43V/F59L/ (0.05) (1.73) (1.60) E77K/P109S/I118T Domain 2:CD86 221 CD28 Q35H/H90L/Q102H Domain 1: CD80 189 PD-L1 3383 4515 5158 90E88D/K89R/D90K/A91G/ (2.75) (1.70) (0.46) (1.30) F92Y/K93R Domain 2:ICOSL 213 ICOS/ N52S/N57Y/H94D/L96F/ CD28 L98F/Q100R/G103E/ F120S Domain1: CD80 193 PD-L1 2230 2148 3860 112 A12T/H18L/N43V/F59L/ (1.81) (0.81)(0.35) (1.62) E77K/P109S/I118T Domain 2: ICOSL 213 ICOS/N52S/N57Y/H94D/L96F/ CD28 L98F/Q100R/G103E/ F120S Domain 1: CD80 193PD-L1 5665 6446 15730 126 A12T/H18L/N43V/F59L/ ICOS/ (4.61) (2.43)(1.41) (1.83) E77K/P109S/I118T CD28 Domain 2: ICOSL 199 N52D Domain 1:CD80 189 PD-L1 6260 4543 11995 269 E88D/K89R/D90K/A91G/ (5.09) (1.71)(1.08) (3.90) F92Y/K93R Domain 2: ICOSL 201 ICOS/ N52H/N57Y/Q100P CD28Domain 1: CD80 193 PD-L1 3359 3874 8541 97 A12T/H18L/N43V/F59L/ (2.73)(1.46) (0.77) (1.41) E77K/P109S/I118T Domain 2: ICOSL 201 ICOS/N52H/N57Y/Q100P CD28 Domain 1: ICOSL WT 196 3000 2966 14366 101 Domain2: CD80 WT 152 (1.00) (1.00) (1.00) (1.00) Domain 1: CD86 WT 220 49461517 125 Domain 2: CD80 WT 152 (1.00) (1.00) (1.00) Domain 1: CD80 192CD28 2832 3672 114 R29H/Y31H/T41G/Y87N/ (1.73) (0.58) (1.16)E88G/K89E/D90N/A91G/ P109S Domain 2: CD80 189 PD-L1 E88D/K89R/D90K/A91G/F92Y/K93R Domain 1: CD80 192 CD28 4542 2878 142 R29H/Y31H/T41G/Y87N/(2.78) (0.45) (1.45) E88G/K89E/D90N/A91G/ P109S Domain 2: CD80 193 PD-L1A12T/H18L/N43V/F59L/ E77K/P109S/I118T Domain 1: CD80 175 CD28 938 995102 I67T/L70Q/A91G/T120S (0.57) (0.16) (1.04) Domain 2: CD80 189 PD-L1E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: CD80 175 CD28 4153 2827 108I67T/L70Q/A91G/T120S Domain 2: CD80 193 PD-L1 (2.54) (0.45) (1.10)A12T/H18L/N43V/F59L/ E77K/P109S/I118T Domain 1: CD86 221 CD28 14608 2535257 Q35H/H90L/Q102H (2.95) (1.67) (2.06) Domain 2: CD80 189 PD-L1E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: CD86 221 CD28 2088 2110 101Q35H/H90L/Q102H (0.42) (1.39) (0.81) Domain 2: CD80 193 PD-L1A12T/H18L/N43V/F59L/ E77K/P109S/I118T Domain 1: ICOSL 213 ICOS/ 36344893 6403 123 N52S/N57Y/H94D/L96F/ CD28 (1.21) (1.65) (0.45) (1.22)L98F/Q100R/G103E/ F120S Domain 2: CD80 189 PD-L1 E88D/K89R/D90K/A91G/F92Y/K93R Domain 1: ICOSL 213 ICOS/ 1095 5929 7923 127N52S/N57Y/H94D/L96F/ CD28 (0.37) (2.0) (0.55) (1.26) L98F/Q100R/G103E/F120S Domain 2: CD80 193 PD-L1 A12T/H18L/N43V/F59L/ E77K/P109S/I118TDomain 1: ICOSL 199 ICOSL/ 2023 5093 16987 125 N52D CD28 (0.67) (1.72)(1.18) (1.24) Domain 2: CD80 189 PD-L1 E88D/K89R/D90K/A91G/ F92Y/K93RDomain 1: ICOSL 199 ICOS/ 3441 3414 20889 165 N52D CD28 (1.15) (1.15)(1.45) (1.63) Domain 2: CD80 193 PD-L1 A12T/H18L/N43V/F59L/E77K/P109S/I118T Domain 1: ICOSL 201 ICOS/ 7835 6634 20779 95N52H/N57Y/Q100P CD28 (2.61) (2.24) (1.45) (0.94) Domain 2: CD80 189PD-L1 E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: ICOSL 201 ICOS/ 8472 378913974 106 N52H/N57Y/Q100P CD28 (2.82) (1.28) (0.97) (1.05) Domain 2:CD80 193 PD-L1 A12T/H18L/N43V/F59L/ E77K/P1098/I118T

TABLE 13 Stacked variant IgV Fc fusion proteins containing two CD80 IgVdomains Counter Binding Activity Functional Domain Structure SEQ IDstructure PD-L1 MFI CTLA-4 MFI Activity MLR N terminal to C terminal: NOselected (WT parental (WT parental IFN-gamma domain 1/domain 2/Fc (IgV)against MFI ratio) MFI ratio) pg/ml Domain 1: CD80 WT 152 6297 469835166 Domain 2: CD80 WT 152 (1.00) (1.00) (1.00) Domain 1: CD80 195CTLA-4 2464 4955 5705 V68M/L70P/L72P/K86E (0.39) (1.05) (0.16) Domain 2:CD80 189 PD-L1 E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: CD80 194 CTLA-41928 1992 1560 R29V/Y31F/K36G/M38L/N43Q/ (0.31) (0.42) (0.04)E81R/V83I/L85I/K89R/D90L/ A91E/F92N/K93Q/R94G Domain 2: CD80 189 PD-L1E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: CD80 195 CTLA-4 1215 1382 2171V68M/L70P/L72P/K86E (0.19) (0.29) (0.06) Domain 2: CD80 193 PD-L1A12T/H18L/N43V/F59L/ E77K/P109S/I118T Domain 1: CD80 194 CTLA-4 15921962 1512 R29V/Y31F/K36G/M38L/N43Q/ (0.25) (0.42) (0.04)E81R/V83I/L85I/K89R/D90L/ A91E/F92N/K93Q/R94G Domain 2: CD80 193 PD-L1A12T/H18L/N43V/F59L/E77K/ P109S/I118T Domain 1: CD80 189 PD-L1 1747 20579739 E88D/K89R/D90K/A91G/ (0.28) (0.44) (0.28) F92Y/K93R Domain 2: CD80195 CTLA-4 V68M/L70P/L72P/K86E Domain 1: CD80 189 PD-L1 1752 1772 5412E88D/K89R/D90K/A91G/ (0.28) (0.38) (0.15) F92Y/K93R Domain 2: CD80 194CTLA-4 R29V/Y31F/K36G/M38L/N43Q/ E81R/V83I/L85I/K89R/D90L/A91E/F92N/K93Q/R94G Domain 1: CD80 193 PD-L1 1636 1887 7608A12T/H18L/N43V/F59L/E77K/ (0.26) (0.40) (0.22) P109S/I118T Domain 2:CD80 195 CTLA-4 V68M/L70P/L72P/K86E Domain 1: CD80 193 PD-L1 2037 482211158 A12T/H18L/N43V/F59L/E77K/ (0.32) (1.03) (0.32) P109S/I118T Domain2: CD80 194 CTLA-4 R29V/Y31F/K36G/M38L/N43Q/ E81R/V83I/L85I/K89R/D90L/A91E/F92N/K93Q/R94G

TABLE 14 Stacked variant IgV Fc fusion proteins containing a CD80 orCD86 IgV domain and an ICOSL IgV domain Counter Binding ActivityFunctional Domain Structure SEQ ID structure PD-L1 MFI CTLA-4 MFIActivity MLR N terminal to C terminal: NO selected (WT parental (WTparental IFN-gamma domain 1/domain 2/Fc (IgV) against MFI ratio) MFIratio) pg/ml Domain 1: CD80 WT 152 1230 11122 1756 Domain 2: ICOSL WT196 (1.00) (1.00) (1.00) Domain 1: CD86 WT 220 29343 55193 6305 Domain2: ICOSL WT 196 (1.00) (1.00) (1.00) Domain 1: CD80 192 CD28 2280 31812281 R29H/Y31H/T41G/Y87N/E88G/ (1.85) (0.29) (1.30) K89E/D90N/A91G/P109SDomain 2: ICOSL 213 ICOS/CD28 N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S Domain 1: CD80 175 CD28 2309 26982 1561I67T/L70Q/A91G/T120S (1.88) (2.43) (0.89) Domain 2: ICOSL 213 ICOS/CD28N52S/N57Y/H94D/L96F/L98F/ Q100R/G103E/F120S Domain 1: CD80 192 CD28 428522744 1612 R29H/Y31H/T41G/Y87N/E88G/ (3.48) (2.04) (0.92)K89E/D90N/A91G/P109S Domain 2: ICOSL 199 ICOS/CD28 N52D Domain 1: CD80175 CD28 3024 16916 3857 I67T/L70Q/A91G/T120S (2.46) (1.52) (2.20)Domain 2: ICOSL 199 ICOS/CD28 N52D Domain 1: CD80 192 CD28 6503 72406886 R29H/Y31H/T41G/Y87N/E88G/ (5.29) (0.65) (3.92) K89E/D90N/A91G/P109SDomain 2: ICOSL 201 ICOS/CD28 N52H/N57Y/Q100P Domain 1: CD80 175 CD283110 4848 3393 I67T/L70Q/A91G/T120S (2.53) (0.44) (1.93) Domain 2: ICOSL201 ICOS/CD28 N52H/N57Y/Q100P Domain 1: CD86 221 CD28 Q35H/H90L/Q102HDomain 2: ICOSL 213 ICOS/CD28 11662 21165 880 N52S/N57Y/H94D/L96F/L98F/(0.40) (0.38) (0.14) Q100R/G103E/F120S Domain 1: CD86 221 CD28 2423073287 1110 Q35H/H90L/Q102H (0.83) (1.33) (0.18) Domain 2: ICOSL 199ICOS/CD28 N52D Domain 1: CD86 221 CD28 1962 1630 587 Q35H/H90L/Q102HICOS/CD28 (0.07) (0.03) (0.09) Domain 2: ICOSL 201 N52H/N57Y/Q100PDomain 1: ICOSL WT 196 3000 14366 4113 Domain 2: CD80 WT 152 (1.00)(1.00) (1.00) Domain 1: ICOSL WT 196 18005 53602 18393 Domain 2: CD86 WT220 (1.00) (1.00) (1.00) Domain 1: ICOSL 213 ICOSL/CD28 10426 5128618680 N52S/N57Y/H94D/L96F/L98F/ (3.48) (3.57) (4.54) Q100R/G103E/F120SDomain 2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/E88G/ K89E/D90N/A91G/P109SDomain 1: ICOSL 213 ICOS/CD28 17751 29790 10637N52S/N57Y/H94D/L96F/L98F/ (5.92) (2.07) (2.59) Q100R/G103E/F120S Domain2: CD80 175 CD28 I67T/L70Q/A91G/T120S Domain 1: ICOSL 199 ICOS/CD28 278825870 6205 N52D (0.93) (1.80) (1.51) Domain 2: CD80 192 CD28R29H/Y31H/T41G/Y87N/E88G/ K89E/D90N/A91G/P109S Domain 1: ICOSL 199ICOS/CD28 2522 13569 5447 N52D (0.84) (0.94) (1.32) Domain 2: CD80 175CD28 I67T/L70Q/A91G/T120S Domain 1: ICOSL 201 ICOS/CD28 N52H/N57Y/Q100PDomain 2: CD80 192 CD28 9701 9187 5690 R29H/Y31H/T41G/Y87N/E88G/ (3.23)(0.64) (1.38) K89E/D90N/A91G/P109S Domain 1: ICOSL 213 ICOS/CD28 2705021257 8131 N52S/N57Y/H94D/L96F/L98F/ (1.50) (0.40) (0.44)Q100R/G103E/F120S Domain 2: CD86 221 CD28 Q35H/H90L/Q102H Domain 1:ICOSL 199 ICOS/CD28 34803 80210 6747 N52D (1.93) (1.50) (0.37) Domain 2:CD86 221 CD28 Q35H/H90L/Q102H Domain 1: ICOSL 201 ICOS/CD28 5948 426826219 N52H/N57Y/Q100P (0.33) (0.08) (1.43) Domain 2: CD86 221 CD28Q35H/H90L/Q102H

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. An immunomodulatory protein that is an Fc fusionprotein comprising a polypeptide operably linked to an Fc domain,wherein the polypeptide is an affinity modified CD80 immunoglobulinsuperfamily (IgSF) domain comprising one or more amino acidsubstitution(s) in a wild-type CD80 IgSF domain, wherein: the wild-typeCD80 IgSF domain is a wild-type CD80 IgV IgSF domain; the affinitymodified CD80 IgSF domain comprises at least 93% sequence identity tothe wild-type CD80 IgV IgSF domain contained in the sequence of aminoacids set forth in SEQ ID NO:1; the affinity modified CD80 IgSF domainhas increased binding to at least two cognate binding partners comparedto the wild-type CD80 IgSF domain, wherein the at least two cognatebinding partners comprise CD28 and PD-L1; and the affinity modified CD80IgSF domain specifically binds non-competitively to the at least twocognate binding partners.
 2. The immunomodulatory protein of claim 1,wherein the immunomodulatory protein is soluble.
 3. The immunomodulatoryprotein of claim 1, wherein the immunomodulatory protein lacks atransmembrane domain and cytoplasmic domain of the wild-type CD80 IgSFdomain.
 4. The immunomodulatory protein of claim 1, wherein the affinitymodified CD80 IgSF domain has at least 120% of the binding affinity asthe wild-type CD80 IgSF domain to each of the at least two cognatebinding partners.
 5. A nucleic acid molecule, encoding theimmunomodulatory protein of claim
 1. 6. The immunomodulatory protein ofclaim 1, wherein the Fc domain is a variant Fc domain with reducedeffector function.
 7. The immunomodulatory protein of claim 1 that is adimer.
 8. The immunomodulatory protein of claim 1, wherein the Fc domainis an Fc of an IgG2 domain or is a variant thereof with reduced effectorfunction.
 9. The immunomodulatory protein of claim 1, wherein the Fcdomain is an Fc of an IgG1 or is a variant thereof with reduced effectorfunction.
 10. A nucleic acid molecule, encoding the immunomodulatoryprotein of claim
 6. 11. The immunomodulatory protein of claim 7 whereinthe dimer is a homodimer.
 12. The immunomodulatory protein of claim 1that is a purified homodimer.